Nematode MDH-like sequences

ABSTRACT

Disclosed are two nucleic acid molecules from  M. incognita  encoding malate dehydrogenase-like (MDH-like) polypeptides. The MDH-like polypeptide sequences are also provided, as are vectors, host cells, and recombinant methods for production of MDH-like nucleotides and polypeptides. The invention further relates to screening methods for identifying inhibitors and/or activators, as well as methods for antibody production.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to provisional application serial No.60/266,037, filed Feb. 2, 2001, the contents of which are herebyincorporated by reference.

BACKGROUND

Nematodes (derived from the Greek word for thread) are active, flexible,elongate, organisms that live on moist surfaces or in liquidenvironments, including films of water within soil and moist tissueswithin other organisms. While only 20,000 species of nematode have beenidentified, it is estimated that 40,000 to 10 million actually exist.Some species of nematodes have evolved as very successful parasites ofboth plants and animals and are responsible for significant economiclosses in agriculture and livestock and for morbidity and mortality inhumans (Whitehead (1998) Plant Nematode Control. CAB International, NewYork).

Nematode parasites of plants can inhabit all parts of plants, includingroots, developing flower buds, leaves, and stems. Plant parasites areclassified on the basis of their feeding habits into the broadcategories: migratory ectoparasites, migratory endoparasites, andsedentary endoparasites. Sedentary endoparasites, which include the rootknot nematodes (Meloidogyne) and cyst nematodes (Globodera andHeterodera) induce feeding sites and establish long-term infectionswithin roots that are often very damaging to crops (Whitehead, supra).It is estimated that parasitic nematodes cost the horticulture andagriculture industries in excess of $78 billion worldwide a year, basedon an estimated average 12% annual loss spread across all major crops.For example, it is estimated that nematodes cause soybean losses ofapproximately $3.2 billion annually worldwide (Barker et al. (1994)Plant and Soil Nematodes: Societal Impact and Focus for the Future. TheCommittee on National Needs and Priorities in Nematology. CooperativeState Research Service, US Department of Agriculture and Society ofNematologists). Several factors make the need for safe and effectivenematode controls urgent. Continuing population growth, famines, andenvironmental degradation have heightened concern for the sustainabilityof agriculture, and new government regulations may prevent or severelyrestrict the use of many available agricultural anthelmintic agents.

The situation is particularly dire for high value crops such asstrawberries and tomatoes where chemicals have been used extensively tocontrol soil pests. The soil fumigant methyl bromide has been usedeffectively to reduce nematode infestations in a variety of thesespecialty crops. It is however regulated under the U.N. MontrealProtocol as an ozone-depleting substance and is scheduled forelimination in 2005 in the US (Carter (2001) California Agriculture,55(3):2). It is expected that strawberry and other commodity cropindustries will be significantly impacted if a suitable replacement formethyl bromide is not found. Presently there are a very small array ofchemicals available to control nematodes and they are frequentlyinadequate, unsuitable, or too costly for some crops or soils (Becker(1999) Agricultural Research Magazine 47(3):22-24; U.S. Pat. Nos.6,048,714). The few available broad-spectrum nematicides such as Telone(a mixture of 1,3-dichloropropene and chloropicrin) have significantrestrictions on their use because of toxicological concerns (Carter(2001) California Agriculture, Vol. 55(3):12-18).

Fatty acids are a class of natural compounds that have been investigatedas alternatives to the toxic, non-specific organophosphate, carbamateand fumigant pesticides (Stadler et al. (1994) Planta Medica60(2):128-132; U.S. Pat. Nos. 5,192,546; 5,346,698; 5,674,897;5,698,592; 6,124,359). It has been suggested that fatty acids derivetheir pesticidal effects by adversely interfering with the nematodecuticle or hypodermis via a detergent (solubilization) effect, orthrough direct interaction of the fatty acids and the lipophilic regionsof target plasma membranes (Davis et al. (1997) Journal of Nematology29(4S):677-684). In view of this general mode of action it is notsurprising that fatty acids are used in a variety of pesticidalapplications including as herbicides (e.g., SCYTHE by Dow Agrosciencesis the C9 saturated fatty acid pelargonic acid), as bactericides andfungicides (U.S. Pat. Nos. 4,771,571; 5,246,716) and as insecticides(e.g., SAFER INSECTICIDAL SOAP by Safer, Inc.).

The phytotoxicity of fatty acids has been a major constraint on theirgeneral use in agricultural applications (U.S. Pat. No. 5,093,124) andthe mitigation of these undesirable effects while preserving pesticidalactivity is a major area of research. The esterification of fatty acidscan significantly decrease their phytotoxicity (U.S. Pat. Nos.5,674,897; 5,698,592; 6,124,359). Such modifications can however lead todramatic loss of nematicidal activity as is seen for linoleic, linolenicand oleic acid (Stadler et al. (1994) Planta Medica 60(2):128-132) andit may be impossible to completely decouple the phytotoxicity andnematicidal activity of pesticidal fatty acids because of theirnon-specific mode of action. Perhaps not surprisingly, the nematicidalfatty acid pelargonic acid methyl ester (U.S. Pat. Nos. 5,674,897;5,698,592; 6,124,359) shows a relatively small “therapeutic window”between the onset of pesticidal activity and the observation ofsignificant phytotoxicity (Davis et al. (1997) J Nematol29(4S):677-684). This is the expected result if both the phytotoxicityand the nematicidial activity derive from the non-specific disruption ofplasma membrane integrity. Similarly the rapid onset of pesticidalactivity seen with many nematicidal fatty acids at therapeuticconcentrations (U.S. Pat. Nos. 5,674,897; 5,698,592; 6,124,359) suggestsa non-specific mechanism of action, possibly related to the disruptionof membranes, action potentials and neuronal activity.

Ricinoleic acid, the major component of castor oil, provides anotherexample of the unexpected effects esterification can have on fatty acidactivity. Ricinoleic acid has been shown to have an inhibitory effect onwater and electrolyte absorption using everted hamster jejunal and ilealsegments (Gaginella et al. (1975) J Pharmacol Exp Ther 195(2):355-61)and to be cytotoxic to isolated intestinal epithelial cells (Gaginellaet al. (1977) J Pharmacol Exp Ther 201(1):259-66). These features arelikely the source of the laxative properties of castor oil which isgiven as a purgative in humans and livestock. In contrast, the methylester of ricinoleic acid is ineffective at suppressing water absorptionin the hamster model (Gaginella et al. (1975) J Pharmacol Exp Ther195(2):355-61). (N.B. Castor oil is a component of some de-wormingprotocols because of its laxative properties.)

The macrocyclic lactones (e.g., avermectins and milbemycins) anddelta-toxins from Bacillus thuringiensis (Bt) are chemicals that inprinciple provide excellent specificity and efficacy and should allowenvironmentally safe control of plant parasitic nematodes.Unfortunately, in practice, these two approaches have proven lesseffective for agricultural applications against root pathogens. Althoughcertain avermectins show exquisite activity against plant parasiticnematodes these chemicals are hampered by poor bioavailability due totheir light sensitivity, degradation by soil microorganisms and tightbinding to soil particles (Lasota & Dybas (1990) Acta Leiden59(1-2):217-225; Wright & Perry (1998) Musculature and Neurobiology. In:The Physiology and Biochemistry of Free-Living and Plant-parasiticNematodes (eds R. N. Perry & D. J. Wright), CAB International 1998).Consequently despite years of research and extensive use against animalparasitic nematodes, mites and insects (plant and animal applications),macrocyclic lactones (e.g., avernectins and milbemycins) have never beencommercially developed to control plant parasitic nematodes in the soil.

Bt delta toxins must be ingested to affect their target organ the brushborder of midgut epithelial cells (Marroquin et al. (2000) Genetics.155(4):1693-1699). Consequently they are not anticipated to be effectiveagainst the dispersal, non-feeding, juvenile stages of plant parasiticnematodes in the field. These juvenile stages only commence feeding whena susceptible host has been infected, thus to be effective nematicidesmay need to penetrate the cuticle. In addition, soil mobility of arelatively large 65-130 kDa protein—the size of typical Bt deltatoxins—is expected to be poor and delivery in planta is likely to beconstrained by the exclusion of large particles by the feeding tube ofcertain plant parasitic nematodes such as Heterodera (Atkinson et al.(1998) Engineering resistance to plant-parasitic nematodes. In: ThePhysiology and Biochemistry of Free-Living and Plant-parasitic Nematodes(eds R. N. Perry & D. J. Wright), CAB International 1998).

Many plant species are known to be highly resistant to nematodes. Themost well documented of these include marigolds (Tagetes spp.),rattlebox (Crotalaria spectabilis), chrysanthemums (Chrysanthemum spp.),castor bean (Ricinus communis), margosa (Azardiracta indica), and manymembers of the family Asteraceae (family Compositae) (Hackney &Dickerson. (1975) J Nematol 7(1):84-90). The active principle(s) forthis nematicidal activity has not been discovered in all of theseexamples and no plant-derived products are sold commercially for controlof nematodes. In the case of the Asteraceae, the photodynamic compoundalpha-terthienyl has been shown to account for the strong nematicidalactivity of the roots. Castor beans are plowed under as a green manurebefore a seed crop is set. However, a significant drawback of the castorplant is that the seed contains toxic compounds (such as ricin) that cankill humans, pets, and livestock and is also highly allergenic.

There remains an urgent need to develop environmentally safe,target-specific ways of controlling plant parasitic nematodes. In thespecialty crop markets, economic hardship resulting from nematodeinfestation is highest in strawberries, bananas, and other high valuevegetables and fruits. In the high-acreage crop markets, nematode damageis greatest in soybeans and cotton. There are however, dozens ofadditional crops that suffer from nematode infestation including potato,pepper, onion, citrus, coffee, sugarcane, greenhouse ornamentals andgolf course turf grasses.

Nematode parasites of vertebrates (e.g., humans, livestock and companionanimals) include gut roundworms, hookworns, pinworms, whipworms, andfilarial worms. They can be transmitted in a variety of ways, includingby water contamination, skin penetration, biting insects, or byingestion of contaminated food.

In domesticated animals, nematode control or “de-worming” is essentialto the economic viability of livestock producers and is a necessary partof veterinary care of companion animals. Parasitic nematodes causemortality in animals (e.g., heartworm in dogs and cats) and morbidity asa result of the parasites' inhibiting the ability of the infected animalto absorb nutrients. The parasite-induced nutrient deficiency results indiseased livestock and companion animals (i.e., pets), as well as instunted growth. For instance, in cattle and dairy herds, a singleuntreated infection with the brown stomach worm can permanently stunt ananimal's ability to effectively convert feed into muscle mass or milk.

Two factors contribute to the need for novel anthelmintics and vaccinesfor control of parasitic nematodes of animals. First, some of the moreprevalent species of parasitic nematodes of livestock are buildingresistance to the anthelmintic drugs available currently, meaning thatthese products will eventually lose their efficacy. These developmentsare not surprising because few effective anthelmintic drugs areavailable and most have been used continuously. Presently a number ofparasitic species has developed resistance to most of the anthelmintics(Geents et al. (1997) Parasitology Today 13:149-151; Prichard (1994)Veterinary Parasitology 54:259-268). The fact that many of theanthelmintic drugs have similar modes of action complicates matters, asthe loss of sensitivity of the parasite to one drug is often accompaniedby side resistance—that is, resistance to other drugs in the same class(Sangster & Gill (1999) Parasitology Today Vol. 15(4):141-146).Secondly, there are some issues with toxicity for the major compoundscurrently available.

Human infections by nematodes result in significant mortality andmorbidity, especially in tropical regions of Africa, Asia, and theAmericas. The World Health Organization estimates 2.9 billion people areinfected with parasitic nematodes. While mortality is rare in proportionto total infections (180,000 deaths annually), morbidity is tremendousand rivals tuberculosis and malaria in disability adjusted life yearmeasurements. Examples of human parasitic nematodes include hookworm,filarial worms, and pinworms. Hookworm is the major cause of anemia inmillions of children, resulting in growth retardation and impairedcognitive development. Filarial worm species invade the lymphatics,resulting in permanently swollen and deformed limbs (elephantiasis) andinvade the eyes causing Aftican Riverblindness. Ascaris lumbricoides,the large gut roundworm infects more than one billion people worldwideand causes malnutrition and obstructive bowl disease. In developedcountries, pinworms are common and often transmitted through children indaycare.

Even in asymptomatic parasitic infections, nematodes can still deprivethe host of valuable nutrients and increase the ability of otherorganisms to establish secondary infections. In some cases, infectionscan cause debilitating illnesses and can result in anemia, diarrhea,dehydration, loss of appetite, or death.

While public health measures have nearly eliminated one tropicalnematode (the water-borne Guinea worm), cases of other worm infectionshave actually increased in recent decades. In these cases, drugintervention provided through foreign donations or purchased by thosewho can afford it remains the major means of control. Because of thehigh rates of reinfection after drug therapy, vaccines remain the besthope for worm control in humans. There are currently no vaccinesavailable.

Until safe and effective vaccines are discovered to prevent parasiticnematode infections, anthelmintic drugs will continue to be used tocontrol and treat nematode parasitic infections in both humans anddomestic animals. Finding effective compounds against parasiticnematodes has been complicated by the fact that the parasites have notbeen amenable to culturing in the laboratory. Parasitic nematodes areoften obligate parasites (i.e., they can only survive in theirrespective hosts, such as in plants, animals, and/or humans) with slowgeneration times. Thus, they are difficult to grow under artificialconditions, making genetic and molecular experimentation difficult orimpossible. To circumvent these limitations, scientists have usedCaenorhabidits elegans as a model system for parasitic nematodediscovery efforts.

C. elegans is a small free-living bacteriovorous nematode that for manyyears has served as an important model system for multicellular animals(Burglin (1998) Int. J. Parasitol., 28(3): 395-411). The genome of C.elegans has been completely sequenced and the nematode shares manygeneral developmental and basic cellular processes with vertebrates(Ruvkin et al. (1998) Science 282: 2033-41). This, together with itsshort generation time and ease of culturing, has made it a model systemof choice for higher eukaryotes (Aboobaker et al. (2000) Ann. Med. 32:23-30).

Although C. elegans serves as a good model system for vertebrates, it isan even better model for study of parasitic nematodes, as C. elegans andother nematodes share unique biological processes not found invertebrates. For example, unlike vertebrates, nematodes produce and usechitin, have gap junctions comprised of innexin rather than connexin andcontain glutamate-gated chloride channels rather than glycine-gatedchloride channels (Bargmann (1998) Science 282: 2028-33). The latterproperty is of particular relevance given that the avermectin class ofdrugs is thought to act at glutamate-gated chloride receptors and ishighly selective for invertebrates (Martin (1997) Vet. J. 154:11-34).

A subset of the genes involved in nematode specific processes will beconserved in nematodes and absent or significantly diverged fromhomologues in other phyla. In other words, it is expected that at leastsome of the genes associated with functions unique to nematodes willhave restricted phylogenetic distributions. The completion of the C.elegans genome project and the growing database of expressed sequencetags (ESTs) from numerous nematodes facilitate identification of these“nematode specific” genes. In addition, conserved genes involved innematode-specific processes are expected to retain the same or verysimilar functions in different nematodes. This functional equivalencehas been demonstrated in some cases by transforming C. elegans withhomologous genes from other nematodes (Kwa et al. (1995) J. Mol. Biol.246:500-10; Redmond et al. (2001) Mol. Biochem. Parasitol. 112:125-131).This sort of data transfer has been shown in cross phyla comparisons forconserved genes and is expected to be more robust among species within aphylum. Consequently, C. elegans and other free-living nematode speciesare likely excellent surrogates for parasitic nematodes with respect toconserved nematode processes.

Many expressed genes in C. elegans and certain genes in otherfree-living nematodes can be “knocked out” genetically by a processreferred to as RNA interference (RNAi), a technique that provides apowerful experimental tool for the study of gene function in nematodes(Fire et al. (1998) Nature 391(6669):806-811; Montgomery et al. (1998)Proc. Natl. Acad. Sci. USA 95(26):15502-15507). Treatment of a nematodewith double-stranded RNA of a selected gene can destroy expressedsequences corresponding to the selected gene thus reducing expression ofthe corresponding protein. By preventing the translation of specificproteins, their functional significance and essentiality to the nematodecan be assessed. Determination of essential genes and theircorresponding proteins using C. elegans as a model system will assist inthe rational design of anti-parasitic nematode control products.

SUMMARY

The invention features nucleic acid molecules encoding M. incognitamalate dehydrogenase (MDH) and other nematode MDH-like polypeptides. M.incognita is a root knot nematode that causes substantial damage tocrops, particularly to cotton, tobacco, pepper, and tomato. In part, theMDH-like nucleic acids and polypeptides of the invention allow for theidentification of a nematode species, and for the identification ofcompounds that bind or alter the activity of MDH-like polypeptides. Suchcompounds may provide a means of combating diseases and infestationscaused by nematodes, particularly by M. incognita, e.g., in tobacco,cotton, pepper or tomato plants.

The invention is based, in part, on the identification of a cDNA(complimentary DNA) encoding M. incognita MDH1 (SEQ ID NO:1). This 1327nucleotide cDNA has a 1098 nucleotide open reading frame (SEQ ID NO:5)encoding a 366 amino acid polypeptide (SEQ ID NO:3). The invention isalso based, in part, on the identification of a cDNA encoding M.incognita MDH2 (SEQ ID NO:2). This 2068 nucleotide cDNA has a 1098nucleotide open reading frame (SEQ ID NO:6) encoding a 366 amino acidpolypeptide (SEQ ID NO:4).

In one aspect, the invention features novel nematode malatedehydrogenase (MDH)-like polypeptides. Such polypeptides includepurified polypeptides having the amino acid sequences set forth in SEQID NO:3 and SEQ ID NO:4. Also included are polypeptides having an aminoacid sequence that is at least about 60%, 70%, 75%, 80%, 85%, 90%, 95%,or 98% identical to SEQ ID NO:3 and/or 4. The purified polypeptides canbe encoded by a nematode gene, e.g., a nematode other than C. elegans.For example, the purified polypeptide have a sequence other than SEQ IDNO:7 or 8 (C. elegans MDH1 and MDH2 respectively). The purifiedpolypeptides can further include a heterologous amino acid sequence,e.g., an amino-terminal or carboxy-terminal sequence derived from adifferent polypeptide. Also featured are purified polypeptide fragmentsof the aforementioned MDH-like polypeptides, e.g., a fragment comprisingat least about 40, 50, 75, 100, 150, 189, 191, 200, 250, 300, or 355amino acids of SEQ ID NO:3; SEQ ID NO:4, or the amino acid sequence ofsome other MDH-like polypeptide other than C. elegans MDH1 or MDH2.Non-limiting examples of such fragments include: fragments comprisingabout amino acid 1 to 149, 1 to 200, 1 to 223, 100 to 223, 149 to 223,149 to 336, 200 to 366, and 223 to 366 of SEQ ID NO:3 and 4. Thepolypeptide or fragment thereof can be modified, e.g., processed,truncated, modified (e.g. by glycosylation, phosphorylation,acetylation, myristylation, prenylation, palmitoylation, amidation,addition of glycerophosphatidyl inositol), or any combination of theabove. Also within the invention are polypeptides consisting of orconsisting essentially of such fragments.

Certain MDH-like polypeptides comprise a sequence of 371 amino acids orfewer.

In another aspect, the invention features novel isolated nucleic acidmolecules encoding a nematode MDH-like polypeptide. Such isolatednucleic acid molecules include nucleic acids having the nucleotidesequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5, or SEQ IDNO:6. Also included are isolated nucleic acid molecules having the samesequence as or encoding the same polypeptide as a nematode MDH-like gene(other than the C. elegans MDH-like genes, MDH1 and MDH2).

Also featured are: 1) isolated nucleic acid molecules having a strandthat hybridizes under low stringency conditions to a single strandedprobe of the sequence of SEQ ID NO: 1 or 2 or their complements and,optionally, encodes a polypeptide of between 280 and 372 amino acids; 2)isolated nucleic acid molecules having a strand that hybridizes underhigh stringency conditions to a single stranded probe of the sequence ofSEQ ID NO: 1 or 2 or their complements and, optionally, encodes apolypeptide of between 280 and 372 amino acids; 3) isolated nucleic acidfragments of MDH-like nucleic acid molecule, e.g., a fragment of SEQ IDNO:1 that is about 573, 575, 750, 1000, 1300, 1500, 1750, or morenucleotides in length or ranges between such lengths, or a fragment ofSEQ ID NO:2 that is about 567, 575, 600, 750, 1000, 1300, 1500, 1750, ormore nucleotides in length or ranges between such lengths; and 4)oligonucleotides that are complementary to an MDH-like nucleic acidmolecule or an MDH-like nucleic acid complement, e.g., anoligonucleotide of about 10, 15, 18, 20, 22, 24, 28, 30, 35, 40, 50, 60,70, 80, or more nucleotides in length. Exemplary oligonucleotides areoligonucleotides which anneal to a site located between a) nucleotidesabout 1 to 84, 61 to 84, 1081 to 1140, 1121 to 1200, 1081 to 1260 or1201 to 1327 of SEQ ID NO:1; or b )nucleotides about 1 to 120,61 to 162,1021 to 1120, 1081 to 1140, 1121 to 1200, 1081 to 1260, or 1081 to 2068of SEQ ID NO:2. Nucleic acid fragments include the followingnon-limiting examples: fragments comprising the sequences of nucleotidesabout 1 to 660, 85 to 660, 660 to 1000, and 1001 to 1367 of SEQ ID NO:1and 2. The isolated nucleic acid can further include a heterologouspromoter operably linked to the MDH-like nucleic acid molecule.

In certain embodiments, the nucleic acid molecule encodes a polypeptidehaving a biological activity of a LMDH1 or MDH2 polypeptide, e.g., theability to interconnect the substrates malate and oxaloacetate.

A molecule featured herein can be from a nematode of the classAraeolaimida, Ascaridida, Chromadorida, Desmodorida, Diplogasterida,Monhysterida, Mononchida, Oxyurida, Rhigonematida, Spirurida, Enoplia,Desmoscolecidae, or Tylenchida, Alternatively, the molecule can be froma species of the class Rhabditida, particularly a species other than C.elegans.

In another aspect, the invention features a vector, e.g., a vectorcontaining an aforementioned nucleic acid. The vector can furtherinclude one or more regulatory elements, e.g., a heterologous promoter.The regulatory elements can be operably linked to the MDH-like nucleicacid molecules in order to express an MDH-like nucleic acid molecule. Inyet another aspect, the invention features a transgenic cell ortransgenic organism having in its genome a transgene containing anaforementioned MDH-like nucleic acid molecule and a heterologous nucleicacid, e.g., a heterologous promoter.

In still another aspect, the invention features an antibody, e.g., anantibody, fragment, or derivative thereof that binds specifically to anaforementioned polypeptide. The specificity of the antibody can be suchthat it does not bind to a C. elegans MDH-like polypeptide. Suchantibodies can be polyclonal or monoclonal antibodies. The antibodiescan be modified, e.g., humanized, rearranged as a single-chain, orCDR-grafted. The antibodies may be directed against a fragment, apeptide, or a discontinuous epitope from an MDH-like polypeptide.

In another aspect, the invention features a method of screening for acompound that binds to a nematode MDH-like polypeptide, e.g., anaforementioned polypeptide. The method includes providing the nematodepolypeptide; contacting the polypeptide with a test compound; anddetecting binding of the test compound to the nematode polypeptide. Inone embodiment, the method further includes contacting a plant ormammalian MDH-like polypeptide with the test compound; and detectingbinding of the test compound to the plant or mammalian MDH-likepolypeptide, e.g., a MDH-like polypeptide of tobacco, cotton, pepper,tomato, or human. A test compound that binds the nematode MDH-likepolypeptide with at least 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or100-fold tighter affinity relative to its affinity for the plant ormammalian MDH-like polypeptide can be identified. In another embodiment,the method further includes contacting nematode MDH-like polypeptidewith the test compound and detecting an MDH-like activity, e.g., theability to introconvert malate and oxaloacetate. A decrease in the levelof MDH-like activity of the polypeptide relative to the level ofMDH-like activity of the polypeptide in the absence of the test compoundis an indication that the test compound is an inhibitor of the MDH-likeactivity. Such inhibitory compounds are potential selective agents forreducing the viability of a nematode expressing an MDH-likepolypeptide., e.g., M. incognita. Preferably, the inhibitory compoundsinhibit the MDH-like activity of both M. incognita MDH1 and MDH2.

Another featured method is a method of screening for a compound thatalters an activity of an MDH-like polypeptide. The method includesproviding the polypeptide; contacting a test compound to thepolypeptide; and detecting an MDH-like activity, wherein a change inMDH-like activity relative to the MDH-like activity of the polypeptidein the absence of the test compound is an indication that the testcompound alters the activity of the polypeptide. The method can furtherinclude contacting the test compound to a plant or mammalian MDH-likepolypeptide; and measuring the MDH-like activity of the plant ormammalian MDH-like polypeptide. A test compound that alters the activityof the nematode MDH-like polypeptide at a given concentration and thatdoes not substantially alter the activity of the plant or mammalianMDH-like polypeptide at the given concentration can be identified. Anadditional method includes screening for both binding to an MDH-likepolypeptide and for alteration in activity of an MDH-like polypeptide.

Yet another featured method is a method of screening for a compound thatalters the viability or fitness of a transgenic cell or organism. Thetransgenic cell or organism has a transgene that expresses an MDH-likepolypeptide. The method includes contacting a test compound to thetransgenic cell or organism; and detecting the viability or fitness ofthe transgenic cell or organism.

Also featured is a method of screening for a compound that alters theexpression of a nematode nucleic acid encoding an MDH-like polypeptide,e.g., a nucleic acid encoding an M. incognita MDH-like polypeptide. Themethod includes contacting a cell, e.g., a nematode cell, with a testcompound, and detecting expression of a nematode nucleic acid encodingan MDH-like polypeptide, e.g., by hybridization to a probe complementaryto the nematode nucleic acid encoding an MDH-like polypeptide. Compoundsidentified by the method are also within the scope of the invention.

In yet another aspect, the invention features a method of treating adisorder caused by a nematode, e.g., M. incognita, in a subject, e.g., ahost plant or host animal. The method includes administering to thesubject an effective amount of an inhibitor of an MDH-like polypeptideactivity or an inhibitor of expression of an MDH-like polypeptide.Non-limiting examples of such inhibitors include: an antisense nucleicacid (or PNA) to an MDH-like nucleic acid, an antibody to an MDH-likepolypeptide, or a small molecule identified as an MDH-like polypeptideinhibitor by a method described herein.

A “purified polypeptide”, as used herein, refers to a polypeptide thathas been separated from other proteins, lipids, and nucleic acids withwhich it is naturally associated. The polypeptide can constitute atleast 10, 20, 50 70, 80 or 95% by dry weight of the purifiedpreparation.

An “isolated nucleic acid” is a nucleic acid, the structure of which isnot identical to that of any naturally-occurring nucleic acid, or tothat of any fragment of a naturally occurring genomic nucleic acidspanning more than three separate genes. The term therefore covers, forexample: (a) a DNA which is part of a naturally occurring genomic DNAmolecule but is not flanked by both of the nucleic acid sequences thatflank that part of the molecule in the genome of the organism in whichit naturally occurs; (b) a nucleic acid incorporated into a vector orinto the genomic DNA of a prokaryote or eukaryote in a manner such thatthe resulting molecule is not identical to any naturally occurringvector or genomic DNA; (c) a separate molecule such as a cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and (d) a recombinant nucleotide sequence that ispart of a hybrid gene, i.e., a gene encoding a fusion protein.Specifically excluded from this definition are nucleic acids present inmixtures of different (i) DNA molecules, (ii) transfected cells, or(iii) cell clones: e.g., as these occur in a DNA library such as a cDNAor genomic DNA library. Isolated nucleic acid molecules according to thepresent invention further include molecules produced synthetically, aswell as any nucleic acids that have been altered chemically and/or thathave modified backbones.

Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid molecule and the phrase “nucleic acid sequence”refers to the sequence of the nucleotides in the nucleic acid molecule,the two phrases can be used interchangeably.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological macromolecules. The substantially pure polypeptide is atleast 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Puritycan be measured by any appropriate standard method, for example, bycolumn chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

The “percent identity” of two amino acid sequences or of two nucleicacids is determined using the algorithm of Karlin and Altschul Proc.Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin andAltschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithmis incorporated into the BLASTN and BLASTX programs (version 2.0) ofAltschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST nucleotidesearches can be performed with the BLASTN program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention. BLAST protein searches can be performedwith the BLASTX program, score=50, wordlength=3 to obtain amino acidsequences homologous to the protein molecules of the invention. Wheregaps exist between two sequences, Gapped BLAST can be utilized asdescribed in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997.When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., BLASTX and BLASTN) can be used. Seehttp://www.ncbi.nlm.nih.gov.

As used herein, the term “transgene” means a nucleic acid sequence(encoding, e.g., one or more subject polypeptides) which is partly orentirely heterologous, i.e., foreign, to the transgenic plant or cellinto which it is introduced or is homologous to an endogenous gene ofthe transgenic plant or cell into which it is introduced, but which isdesigned to be inserted or is inserted into the plant's genome in such away as to alter the genome of the cell into which it is inserted (e.g.,it is inserted at a location which differs from that of the natural geneor its insertion results in a knockout). A transgene can include one ormore transcriptional regulatory sequences and any other nucleic acid,such as introns, that may be necessary for optimal expression of theselected nucleic acid, all operably linked to the selected nucleic acid,and may include an enhancer sequence.

As used herein, the term “transgenic cell” refers to a cell containing atransgene.

As used herein, a “transgenic plant” is any plant in which one or more,or all, of the cells of the plant includes a transgene. The transgenecan be introduced into the cell, directly or indirectly by introductioninto a precursor of the cell, by way of deliberate genetic manipulation,such as by T-DNA mediated transfer, electroporation, or protoplasttransformation. The transgene may be integrated within a chromosome, orit may be extrachromosomally replicating DNA.

As used herein, the term “tissue-specific promoter” means a DNA sequencethat serves as a promoter, i.e., regulates expression of a selected DNAsequence operably linked to the promoter, and which effects expressionof the selected DNA sequence in specific cells of a tissue, such as aleaf, a root, or a stem.

As used herein, the terms “hybridizes under stringent conditions” and“hybridizes under high stringency conditions” refers to conditions forhybridization in 6×sodium chloride/sodium citrate (SSC) at about 45□C.,followed by two washes in 0.2×SSC, 0.1% SDS at 65□C. As used herein, theterm “hybridizes under low stringency conditions” refers to conditionsfor hybridization in 6×sodium chloride/sodium citrate (SSC) at about45□C, followed by two washes in 6×SSC buffer, 0. 1% (w/v) SDS at 2□C.

A “heterologous promoter”, when operably linked to a nucleic acidsequence, refers to a promoter which is not naturally associated withthe nucleic acid sequence.

As used herein, an agent with “anthelmintic activity” is an agent which,when tested, has measurable nematode-killing activity or results ininfertility or sterility in the nematodes such that inviable or nooffspring result. In the assay, the agent is combined with nematodes,e.g., in a well of microtiter dish having agar media containing theagent. Staged adult nematodes are placed on the media The time ofsurvival, viability of offspring, and/or the movement of the nematodesare measured. An agent with “anthelmintic activity” reduces the survivaltime of adult nematodes relative to unexposed similarly-staged adults,e.g., by about 20%, 40%, 60%, 80%, or more. In the alternative, an agentwith “anthelmintic activity” may also cause the nematodes to ceasereplicating, regenerating, and/or producing viable progeny, e.g., byabout 20%, 40%, 60%, 80%, or more.

As used herein, the term “binding” refers to the ability of a firstcompound and a second compound that are not covalently attached tophysically interact. The apparent dissociation constant for a bindingevent can be 1 mM or less, for example, 10 nM, 1 nM, 0.1 nM or less.

As used herein, the term “binds specifically” refers to the ability ofan antibody to discriminate between a target ligand and a non-targetligand such that the antibody binds to the target ligand and not to thenon-target ligand when simultaneously exposed to both the given ligandand non-target ligand, and when the target ligand and the non-targetligand are both present in a molar excess over the antibody.

A used herein, the term “altering an activity” refers to a change inlevel, either an increase or a decrease in the activity, particularly anMDH-like or MDH activity. The change can be detected in a qualitative orquantitative observation. If a quantitative observation is made, and ifa comprehensive analysis is performed over a plurality of observations,one skilled in the art can apply routine statistical analysis toidentify modulations where a level is changed and where the statisticalparameter, the p value, is less than 0.05.

In part, the nematode MDH proteins and nucleic acids described hereinare novel targets for anti-nematode vaccines, pesticides, and drugs.Inhibition of these molecules can provide means of inhibiting nematodemetabolism and/or the nematode life-cycle.

All publications, patents, patent applications and other referencematerials mentioned herein are incorporated by reference in theirentirety.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a listing of a M. incognita MDH-like nucleic acid sequence,MDH1, SEQ ID NO:1, its corresponding encoded amino acid sequence, SEQ IDNO:3 and its open reading frame, SEQ ID NO:5.

FIGS. 2A-B is a listing of a M. incognita MDH-like nucleic acidsequence, MDH2, SEQ ID NO:2, its corresponding encoded amino acidsequence, SEQ ID NO:4, and its open reading frame, SEQ ID NO:6.

FIG. 3 is an alignment of the sequences of C. elegans and M. incognitaMDH-like polypeptides. The sequence labeled “1” is M. incognita MDH1(SEQ ID NO:3). The sequence labeled “2” is M. incognita MDH2 (SEQ IDNO:4). The sequence labeled “3” is C. elegans MDH1 (SEQ ID NO:7). Thesequence labeled “4” is C. elegans MDH2 (SEQ ID NO:8).

DETAILED DESCRIPTION

Malate dehydrogenase (MDH) is a tricarboxylic acid (TCA) cycle enzymethat (i) reduces oxaloacetate to malate with concomitant oxidation ofNADH to NAD and (ii) catalyzes the reverse reaction. The TCA cyclefollows glycolysis and is the convergence point of carbohydrate,protein, and lipid catabolism during aerobic respiration (via acetylCoA). Malate dehydrogenase is also an important component of thebi-directional malate-aspartate shuttle that transports NADH/NAD⁺ intoand out of mitochondria There are two forms of the enzyme, onecytosolic, and the other present in the mitochondrial matrix. Moreover,in certain nematode parasites such as Ascaris, several steps in the TCApathway can be modified, e.g., to run in reverse (Barrett (1981)Biochemistry of Parasitic Helminths. Macmillan, London. 309).

Compounds which inhibit glycolysis can be toxic to nematodes(Butterworth et al. (1989). Revue de Nematologie. 12:63-67).Accordingly, inhibitors of TCA cycle enzymes such a MDH are alsoexpected to be toxic to nematodes. The malate dehydrogenase class ofenzymes include enzymes that are fundamental to tricarboxylic acid (TCA)metabolism. Thus, MDH is an attractive target for the development ofcompounds toxic to nematodes.

The present invention provides nucleic acids from nematodes encodingmalate dehydrogenase (MDH)-like polypeptides. The nucleic acid molecules(SEQ ID NO: 1 and SEQ: ID NO:2) and the encoded malatedehydrogenase-like polypeptides (SEQ ID NO:3 and SEQ ID NO:4) arerecited in FIG. 1 and FIG. 2. The invention is based, in part, on thediscovery of these MDH-like sequences from M. incognita. The followingexample is, therefore, to be construed as merely illustrative, and notlimitative of the remainder of the disclosure in any way whatsoever. Allof the publications cited herein are hereby incorporated by reference intheir entirety.

EXAMPLE

TBLASTN searches identified several expressed sequence tags (ESTs; shortnucleic acid fragment sequences from single sequencing reads) that aresimilar to two MDH-like C. elegans genes, MDH1 (GenBank® accessionnumber T20396; GI:7500583) and MDH2 (GenBank® accession number T18570;GI:7511561). A query with C. elegans MDH1-like sequences identified ESTsin two nematode species: GenBank® accession number AW588457 (GI:7275489)(from Ancylstoma caninum; McCarter et al. (1999) The WashingtonUniversity Nematode EST Project; on the World Wide Web atgenome.wustl.edu/gsc/) similar to C. elegans MDH1 codons 222-364; andGenBank® accession number AW827742 (GI:7921523) (from M. incognita;McCarter et al. (1 999) The Washington University Nematode EST Project;on the World Wide Web at genome.wustl.edu/gsc/) similar to C. elegansMDH1 codons 11-201.

A query with C. elegans MDH2 identified ESTs in three species: GenBank®accession number RO5238 (GI:754974) (from Caenorhabditis briggsae;Hillier et al. (1995) Washington University Caenorhabditis briggsae ESTproject; on the World Wide Web at genome.wustl.edu/gsc/) similar to C.elegans MDH2 codons 292-400; GenBank® accession number AW828934(GI:7922734) (from M. incognita; McCarter et al. (1999) The WashingtonUniversity Nematode EST Project; on the World Wide Web atgenome.wustl.edu/gsc/) similar to C. elegans MDH2 codons 46 to 234; andGenBank® accession number BE581385 (GI:9832327) (from Strongyloidesstercoralis McCarter et al. (1999) The Washington University NematodeEST Project; on the World Wide Web at genome.wustl.edu/gsc/) similar toC. elegans MDH2 codons 48 to 215.

Full-Length MDH-like cDNA Sequences

Full-length (containing the complete open reading frame) sequenceinformation for C. elegans MDH 1 and MDH2 was generated by a computerprogram from raw cosmid sequence data present in the GenBank NRdatabase. Hypothetical exon sequences were spliced together manually byremoving intervening intron sequences such that the final predicted cDNAsequence, when conceptually translated, exactly matched the predictedprotein sequences for C. elegans MDH1 and MDH2 reported in the GenBankNR database.

Plasmid clones, Div109 and Div116, corresponding to the M. incognita ESTsequences GenBank® accession number AW827742 (GI:7921523) and Genbank®accession number AW828934 (GI:7922734) respectively, were obtained fromthe Genome Sequencing Center (St. Louis, Mo.). The cDNA inserts of thetwo plasmids were sequenced in their entirety to obtain the full-lengthsequences of M. incognita MDH1 and MDH2 (SEQ. ID. NO. 1 and 2). Unlessotherwise indicated, all nucleotide sequences determined herein weresequenced with an automated DNA sequencer (such as Model 373 fromApplied Biosystems, Inc.) using processes well-known to those skilled inthe art. The primers in Table 1 were used for sequencing.

TABLE 1 Name Sequence Description T7 5′ gta ata cga ctc act ata ggg c 3′(SEQ ID NO:9) vector poly- linker primer T3 5′ aat taa ccc tca cta aaggg 3′ (SEQ ID NO:10) vector poly- linker primer Oligo 5′ gag aga gag agagag aga gaa cta gtc tcg agt ttt Universal prim- dT ttt ttt ttt ttt tt 3′(SEQ ID NO:11) er to poly A tail Mdh2 5′ agc aac aaa aca ttg gcc 3′ (SEQID NO:12) Mi MDH1 (co- dons 280-285) Mdh3 5′ ggc act gct tgg gtt gat 3′(SEQ ID NO:13) Mi MDH2 (codons 83-88) Mdh4 5′ atc aac cca agc agt gcc 3′(SEQ ID NO:14) Mi MDH2 (codons 83-88) Mdh5 5′ cga tta tgg ggg caa tcacac 3′ (SEQ ID NO:15) Mi MDH2 (co- dons 268-274)

Characterization of M. incognita MDH1 and MDH2

The sequences of the two M. incognita MDH-like nucleic acid moleculesare recited in FIG. 1 and FIG. 2 as SEQ ID NO:1 and SEQ ID NO:2. Boththese nucleotide sequences contain open reading frames encoding a 366amino acid polypeptide. The proteins (having amino acid sequences SEQ IDNO:3 and SEQ ID NO:4, respectively), designated M. incognita MDH1 andMDH2, are approximately 48% and 50% identical to the corresponding C.elegans MDH proteins.

The similarity between M. incognita MDH sequences and other sequenceswas investigated by comparison to the sequence of other nematode genes,such as C. elegans. The similarity between MDH-like proteins from M.incognita and from C. elegans is presented as a multiple alignmentgenerated by the ClustalX multiple alignment program as described below(FIG. 3).

The similarity between M. incognita MDH sequences and other sequenceswas also investigated by comparison to sequence databases using BLASTXanalysis against nr (a non-redundant sequence database available on theWorld Wide Web at ncbi.nlm.nih.gov/) and TBLASTX analysis against dbest(an EST sequence database available on the World Wide Web atncbi.nlm.nih.gov/;top 500 hits; E=1e−4). The “Expect (E) value” is thenumber of sequences that are predicted to align by chance given the sizeof the queried database. This analysis was used to determine thepotential number of plant and vertebrate homologs. Neither C. elegansnor M. incognita (SEQ ID NO:1 and SEQ ID NO:2) MDH 1 nor MDH2-likesequences had plant or vertebrate hits in nr having sufficientsimilarity to meet the threshold E value of 1e−4 (this E valueapproximately corresponds to a threshold for removing sequences having asequence identity of less than about 20% over greater than 50 aminoacids). Three vertebrate hits and one plant hit were found in dbest ashaving weak identity (e.g., less than 35% identity over 100 amino acids)to MDH1 from C. elegans. Thus, the M. incognita MDH -like enzymes ofthis invention do not appear to share significant sequence similaritywith plant or vertebrate forms of the enzyme such as the Homo sapiensmalate dehydrogenase genes P40926 (mitochondrial precursor) and P40925(cytoplasmic form). On the basis of this lack of similarity, theM.incognita MDH-like enzymes are useful targets of inhibitory compoundsselective for nematodes over their hosts (e.g., plants).

Functional predictions were made with the Interpro web server (availableat ebi.ac.uk/interpro/), which provides access to the PRO SITE, BLOCKS,PRINTS and Pfam prediction servers. No hits were found in searches usingeither the C. elegans or M. incognita polypeptides as queries suggestingthat these polypeptides are a novel class of MDH-like enzymes. Proteinlocalization was predicted using the TargetP web server (available atcbs.dtu.dk/services/TargetP/). The C. elegans MDH1 and M. incognita MDH1and MDH 2 (SEQ ID NO:1 and SEQ: ID NO:2) polypeptides were predicted tobe cytoplasmic whereas the C. elegans MDH2 polypeptide was predicted tolocalize to mitochondria.

Identification of Additional MDH-Like Sequences

A skilled artisan can utilize the methods provided in the example aboveto identify additional nematode MDH-like sequences, e.g., MDH-likesequence from nematodes other than C. elegans and M. incognita. Inaddition, nematode MDH-like sequences can be identified by a variety ofmethods including computer-based database searches, hybridization-basedmethods, and functional complementation.

Database Identification. A nematode MDH-like sequence can be identifiedfrom a sequence database, e.g., a protein or nucleic acid database usinga sequence disclosed herein as a query. Sequence comparison programs canbe used to compare and analyze the nucleotide or amino acid sequences.One such software package is the BLAST suite of programs from theNational Center for Biotechnology Institute's (NCBI; Altschul, et al.,(1997) Nuc. Acids Research 25:3389-3402.). An MDH-like sequence of theinvention can be used to query a sequence database, such as nr, nemnoele(a database of nematode sequences extracted from dbest and lacking C.elegans sequences), dbest (expressed sequence tag (EST) sequences), andhtgs (high-throughput genome sequences), using a computer-based search,e.g., FASTA, BLAST, or PSI-BLAST search. Homologous sequences in otherspecies (e.g., humans, plants, animals, fungi) can be detected in aPSI-BLAST search of a database such as nr (E value=1e−2, H. Value=1e−4,using, for example, four iterations; available at ncbi.nlm.nih.gov/).Sequences so obtained can be used to construct a multiple alignment,e.g., a ClustalX alignment, and/or to build a phylogenetic tree, e.g.,in ClustalX using the Neighbor-Joining method (Saitou and Nei, (1987)Mol. Biol. Evol. 4:406-425) and bootstrapping (1000 replicates;Felsenstein, (1985) Evolution 39:783-791). Distances may be correctedfor the occurrence of multiple substitutions [D_(corr)=−1n(1−D−D²/5)where D is the fraction of amino acid differences between two sequences](Kimura (1983). The Neutral Theory of Molecular Evolution).

The aforementioned search strategy can be used to identify MDH-likesequences in nematodes of the following non-limiting, exemplary genera:

Plant nematode genera: Afrina, Anguina, Aphelenchoides, Belonolaimus,Bursaphelenchus, Cacopaurus, Cactodera, Criconema, Criconemoides,Cryphodera, Ditylenchus, Dolichodorus, Dorylaimus, Globodera,Helicotylenchus, Hemicriconemoides, Hemicycliophora, Heterodera,Hirschmanniella, Hoplolaimus, Hypsoperine, Longidorus, Meloidogyne,Mesoanguina, Macobbus, Nacobbodera, Panagrellus, Paratrichodorus,Paratylenchus, Pratylenchus, Pterotylenchus, Punctodera, Radopholus,Rhadinaphelenchus, Rotylenchulus, Rotylenchus, Scutellonema, Subanguina,Thecavermiculatus, Trichodorus, Turbatrix, Tylenchorhynchus,Tylenchulus, Xiphinema.

Animal and human nematode genera: Acanthocheilonema, Aelurostrongylus,Ancylostoma, Angiostrongylus, Anisakis, Ascaris, Ascarops, Bunostomum,Brugia, Capillaria, Chabertia, Cooperia, Crenosoma, Cyathostome species(Small Strongyles), Dictyocaulus, Dioctophyma, Dipetalonema,Dirofiliaria, Dracunculus, Draschia, Elaneophora, Enterobius,Filaroides, Gnathostoma, Gonylonema, Habronema, Haemonchus,Hyostrongylus, Lagochilascaris, Litomosoides, Loa, Mammomonogamus,Mansonella, Muellerius, Metastrongylid, Necator, Nematodirus,Nippostrongylus, Oesophagostomum, Ollulanus, Onchocerca, Ostertagia,Oxyspirura, Oxyuris, Parafilaria, Parascaris, Parastrongyloides,Parelaphostrongylus, Physaloptera, Physocephalus, Protostrongylus,Pseudoterranova, Setaria, Spirocerca, Stephanurus, StephanofilariaStrongyloides, Strongylus, Spirocerca, Syngamus, Teladorsagia, Thelazia,Toxascaris, Toxocara, Trichinella, Trichostrongylus, Trichuris,Uncinaria, and Wuchereria.

Particularly preferred nematode genera include: Plant: Anguina,Aphelenchoides, Belonolaimus, Bursaphelenchus, Ditylenchus,Dolichodorus, Globodera, Heterodera, Hoplolaimus, Longidorus,Meloidogyne, Nacobbus, Pratylenchus, Radopholus, Rotylenchus,Tylenchulus, Xiphinema.

Animal and human: Ancylostoma, Ascaris, Brugia, Capillaria, Cooperia,Cyathostome species, Dictyocaulus, Dirofiliaria, Dracunculus,Enterobius, Haemonchus, Necator, Nematodirus, Oesophagostomum,Onchocerca, Ostertagia, Oxyspirura, Oxyuris, Parascaris, Strongyloides,Strongylus, Syngamus, Teladorsagia, Thelazia, Toxocara, Trichinella,Trichostrongylus, Trichuris, and Wuchereria.

Particularly preferred nematode species include: Plant: Anguina tritici,Aphelenchoides fragariae, Belonolaimus longicaudatus, Bursaphelenchusxylophilus, Ditylenchus destructor, Ditylenchus dipsaci Dolichodorusheterocephalous, Globodera pallida, Globodera rostochiensis, Globoderatabacum, Heterodera avenae, Heterodera cardiolata, Heterodera carotae,Heterodera cruciferae, Heterodera glycines, Heterodera major, Heteroderaschachtii, Heterodera zeae, Hoplolaimus tylenchiformis, Longidorussylphus, Meloidogyne acronea, Meloidogyne arenaria, Meloidogynechitwoodi, Meloidogyne exigua, Meloidogyne graminicola, Meloidogynehapla, Meloidogyne incognita, Meloidogyne javanica, Meloidogyne nassi,Nacobbus batatiformis, Pratylenchus brachyurus, Pratylenchus coffeae,Pratylenchus penetrans, Pratylenchus scribneri, Pratylenchus zeae,Radopholus similis, Rotylenchus reniformis, Tylenchulus semipenetrans,Xiphinema americanum.

Animal and human: Ancylostoma braziliense, Ancylostoma caninum,Ancylostoma ceylanicum, Ancylostoma duodenale, Ancylostoma tubaeforme,Ascaris suum, Ascaris lumbrichoides, Brugia malayi, Capillaria bovis,Capillariaplica, Capillaria feliscati, Cooperia oncophora, Cooperiapunctata, Cyathostome species, Dictyocaulus filaria, Dictyocaulusviviparus, Dictyocaulus arnfieldi, Dirofiliaria immitis, Dracunculusinsignis, Enterobius vermicularis, Haemonchus contortus, Haemonchusplacei, Necator americanus, Nematodirus helvetianus, Oesophagostomumradiatum, Onchocerca volvulus, Onchocerca cervicalis, Ostertagiaostertagi, Ostertagia circumcincta, Oxyuris equi, Parascaris equorum,Strongyloides stercoralis, Strongylus vulgaris, Strongylus edentatus,Syngamus trachea, Teladorsagia circumcincta, Toxocara cati, Trichinellaspiralis, Trichostrongylus axei, Trichostrongylus colubriformis,Trichuris vulpis, Trichuris suis, Trichurs trichiura, and Wuchereriabancrofti.

Further, an MDH-like sequence can be used to identify additionalMDH-like sequence homologs within a genome. Multiple homologous copiesof an MDH-like sequence can be present. For example, a nematode MDH-likesequence can be used as a seed sequence in an iterative PSI-BLAST search(default parameters, substitution matrix=Blosum62, gap open=11, gapextend=1) of a database, such as nr or wormpep (E value=1e−2, Hvalue=1e−4, using, for example 4 iterations) to determine the number ofhomologs in a database, e.g., in a database recording the genome of theorganism (such as in the completed C. elegans genome). A nematodeMDH-like sequence can be present in a genome along with 1, 2, 3, 4, 5,6, 8, 10, or more homologs.

Hybridization Methods. A nematode MDH-like sequence can be identified bya hybridization-based method using a sequence provided herein as aprobe. For example, a library of nematode genomic or cDNA clones can behybridized under low stringency conditions with the probe nucleic acid.Stringency conditions can be modulated to reduce background signal andincrease signal from potential positives. Clones so identified can besequenced to verify that they encode MDH-like sequences.

Another hybridization-based method utilizes an amplification reaction(e.g., the polymerase chain reaction (PCR)). Oligonucleotides, e.g.,degenerate oligonucleotides, are designed to hybridize to a conservedregion of an MDH-like sequence (e.g., a region conserved in all foursequences depicted in FIG. 3). The oligonucleotides are used as primersto amplify an MDH-like sequence from template nucleic acid from anematode, e.g., a nematode other than C. elegans or M. incognita. Theamplified fragment can be cloned and/or sequenced.

Complementation Methods. A nematode MDH-like sequence can be identifiedfrom a complementation screen for a nucleic acid molecule which providesan MDH-like activity to a cell lacking an MDH-like activity. Routinemethods can be used to construct E. coli or yeast strains that lack,e.g., conditionally, MDH activity. For example, an S. cerevisiae straindeleted for all three of the genes, MDH1, MDH2, and MDH3 can begenerated (see, e.g., Steffan and McAlister-Henn (1992) J Biol Chem267:24708-15). Such strains can be transformed with a plasmid libraryexpressing nematode cDNAs. Strains are identified in which MDH activityis restored For example, the mdh1⁻2⁻3⁻ S. cerevisiae strain transformedwith the plasmid library can be grown on acetate to select for strainsexpressing a nematode MDH-like gene. The plasmid harbored by the straincan be recovered to identify and/or characterized the inserted nematodecDNA that provides MDH-like activity when expressed.

Full-length cDNA and Sequencing Methods. The following methods can beused, e.g., alone or in combination with another method describedherein, to obtain a fill-length nematode MDH-like gene and determine itssequence.

Plant parasitic nematodes are maintained on greenhouse pot culturesdepending on nematode preference. Root Knot Nematodes (Meloidogyne sp)are propagated on Rutgers tomato (Burpee). Total RNA is isolated usingthe TRIZOL reagent (Gibco BRL). Briefly, 2 ml of packed worms arecombined with 8 ml TRIZOL reagent and solubilized by vortexing.Following 5 minutes of incubation at room temperature, the samples arespun at 14,000×g for 10 minutes at 4° C. to remove insoluble material.The liquid phase is extracted with 200 μl of chloroform, and the upperaqueous phase is removed to a fresh tube. The RNA is precipitated by theaddition of 500 μl of isopropanol and centrifuged to pellet. The aqueousphase is carefully removed, and the pellet is washed in 75% ethanol andspun to re-collect the RNA pellet. The supernatant is carefully removed,and the pellet is air dried for 10 minutes. The RNA pellet isresuspended in 50 μl of DEPC-H₂0 and analyzed by spectrophotometry atλ260 and 280 nm to determine yield and purity. Yields can be 1-4 mg oftotal RNA from 2 ml of packed worms.

Full-length cDNAs can be generated by using 5′ and 3′ RACE techniques incombination with EST sequence information. The molecular technique 5′RACE (Life Technologies, Inc., Rockville, Md.) is employed to obtaincomplete or near-complete 5′ ends of cDNA sequences for a nematodeMDH-like cDNA sequences. Briefly, following the instructions provided byLife Technologies, first strand cDNA is synthesized from total M.incognita RNA using Murine Leukemia Virus Reverse Transcriptase (M-MLVRT) and a gene specific “antisense” primer, e.g., designed fromavailable EST sequence. RNase H is used to degrade the original MRNAtemplate. The first strand cDNA is separated from unincorporated dNTPs,primers, and proteins using a GlassMAX Spin Cartridge. Terminaldeoxynucleotidyl transferase (TdT) is used to generate a homopolymericdC tailed extension by the sequential addition of dCTP nucleotides tothe 3′ end of the first strand cDNA. Following addition of the dChomopolymeric extension, the first strand cDNA is directly amplifiedwithout further purification using Taq DNA polymerase, a gene specific“antisense” primer designed from available EST sequences to anneal to asite located within the first strand cDNA molecule, and adeoxyinosine-containing primer that anneals to the homopolymeric dCtailed region of the CDNA in a polymerase chain reaction (PCR). 5′ RACEPCR amplification products are cloned into a suitable vector for furtheranalysis and sequenced.

The molecular technique, 3′ RACE (Life Technologies, Inc., Rockville,Md.), is employed to obtain complete or near-complete 3′ ends of cDNAsequences for M. incognita MDH-like cDNA sequences. Briefly, followingthe instructions provided by Life Technologies (Rockville, Md.), firststrand cDNA synthesis is performed on total nematode RNA usingSuperScript™ Reverse Transcriptase and an oligo-dT primer which annealsto the polyA tail. Following degradation of the original mRNA templatewith RNase H, the first strand cDNA is directly PCR amplified withoutfurther purification using Taq DNA polymerase, a gene specific primerdesigned from available EST sequences to anneal to a site located withinthe first strand cDNA molecule, and a “universal” primer which containssequence identity to 5′ end of the oligo-dT primer. 3′ RACE PCRamplification products are cloned into a suitable vector for furtheranalysis and sequenced.

Nucleic Acid Variants

Isolated nucleic acid molecules of the present invention include nucleicacid molecules that have an open reading frame encoding an MDH-likepolypeptide. Such nucleic acid molecules include molecules having: thesequences recited in SEQ ID NO:1 and SEQ ID NO:2; and sequences codingfor the MDH-like proteins recited in SEQ ID NO:3 and SEQ ID NO:4. Thesenucleic acid molecules can be used, for example, in an hybridizationassay to detect the presence of an M. incognita nucleic acid in asample.

The present invention includes nucleic acid molecules such as thoseshown in SEQ ID NO:1 and SEQ ID NO:2 that may be subjected tomutagenesis to produce single or multiple nucleotide substitutions,deletions, or insertions. Nucleotide insertional derivatives of thenematode gene of the present invention include 5′ and 3′ terminalfusions as well as intra-sequence insertions of single or multiplenucleotides. Insertional nucleotide sequence variants are those in whichone or more nucleotides are introduced into a predetermined site in thenucleotide sequence, although random insertion is also possible withsuitable screening of the resulting product. Deletion variants arecharacterized by the removal of one or more nucleotides from thesequence. Nucleotide substitution variants are those in which at leastone nucleotide in the sequence has been removed and a differentnucleotide inserted in its place. Such a substitution may be silent(e.g., synonymous), meaning that the substitution does not alter theamino acid defined by the codon. Alternatively, substitutions aredesigned to alter one amino acid for another amino acid (e.g.,non-synonymous). A non-synonymous substitution can be conservative ornon-conservative. A substitution can be such that activity, e.g., amalate dehydrogenase-like activity, is not impaired. A conservativeamino acid substitution results in the alteration of an amino acid for asimilar acting amino acid, or amino acid of like charge, polarity, orhydrophobicity, e.g., an amino acid substitution listed in Table 2below. At some positions, even conservative amino acid substitutions candisrupt the activity of the polypeptide.

TABLE 2 Conservative Amino Acid Replacements For Amino Acid Code Replacewith any of . . . Alanine Ala Gly, Cys, Ser Arginine Arg Lys, HisAsparagine Asn Asp, Glu, Gln, Aspartic Acid Asp Asn, Glu, Gln CysteineCys Met, Thr, Ser Glutamine Gln Asn, Glu, Asp Glutamic Acid Glu Asp,Asn, Gln Glycine Gly Ala Histidine His Lys, Arg Isoleucine Ile Val, Leu,Met Leucine Leu Val, Ile, Met Lysine Lys Arg, His Methionine Met Ile,Leu, Val Phenylalanine Phe Tyr, His, Trp Proline Pro Serine Ser Thr,Cys, Ala Threonine Thr Ser, Met, Val Tryptophan Trp Phe, Tyr TyrosineTyr Phe, His Valine Val Leu, Ile, Met

The current invention also embodies splice variants of nematode MDH-likesequences.

Another aspect of the present invention embodies a polypeptide-encodingnucleic acid molecule that is capable of hybridizing under conditions oflow stringency to the nucleic acid molecules put forth in SEQ ID NO:1and SEQ ID NO:2, or their complements.

The nucleic acid molecules that encode for MDH-like polypeptides maycorrespond to the naturally-occurring nucleic acid molecules or maydiffer by one or more nucleotide substitutions, deletions, and/oradditions. Thus, the present invention extends to genes and anyfunctional mutants, derivatives, parts, fragments, homologs or analogsthereof or non-functional molecules. Such nucleic acid molecules can beused to detect polymorphisms of MDH genes or MDH-like genes, e.g., inother nematodes. As mentioned below, such molecules are useful asgenetic probes; primer sequences in the enzymatic or chemical synthesisof the gene; or in the generation of immunologically interactiverecombinant molecules. Using the information provided herein, such asthe nucleotide sequence SEQ ID NO:1 or SEQ ID NO:2, a nucleic acidmolecule encoding a MDH-like molecule may be obtained using standardcloning and a screening techniques, such as a method described herein.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as MRNA, or in the form of DNA, including, for example, cDNAand genomic DNA obtained by cloning or produced synthetically. The DNAmay be double-stranded or single-stranded. The nucleic acids may in theform of RNA/DNA hybrids. Single-stranded DNA or RNA can be the codingstrand, also referred to as the sense strand, or the non-coding strand,also known as the anti-sense strand.

One embodiment of the present invention includes a recombinant nucleicacid molecule, which includes at least one isolated nucleic acidmolecule depicted in SEQ ID NO:1 and SEQ ID NO:2, inserted in a vectorcapable of delivering and maintaining the nucleic acid molecule into acell. The DNA molecule may be inserted into an autonomously replicatingfactor (suitable vectors include, for example, pGEM3Z and pcDNA3, andderivatives thereof). The vector nucleic acid may be a bacteriophage DNAsuch as bacteriophage lambda or M13 and derivatives thereof. The vectormay be either RNA or DNA, single- or double-stranded, eitherprokaryotic, eukaryotic, or viral. Vectors can include transposons,viral vectors, episomes, (e.g. plasmids), chromosomes inserts, andartificial chromosomes (e.g. BACs or YACs). Construction of a vectorcontaining a nucleic acid described herein can be followed bytransformation of a host cell such as a bacterium. Suitable bacterialhosts include, but are not limited to, E. coli. Suitable eukaryotichosts include yeast such as Saccharomyces cerevisiae, other fungi,vertebrate cells, invertebrate cells (e.g., insect cells), plant cells,human cells, human tissue cells, and whole eukaryotic organisms. (e.g.,a transgenic plant or a transgenic animal). Further, the vector nucleicacid can be used to generate a virus such as vaccinia or baculovirus.

The present invention also extends to genetic constructs designed forpolypeptide expression. Generally, the genetic construct also includes,in addition to the encoding nucleic acid molecule, elements that allowexpression, such as a promoter and regulatory sequences. The expressionvectors may contain transcriptional control sequences that controltranscriptional initiation, such as promoter, enhancer, operator, andrepressor sequences. A variety of transcriptional control sequences arewell known to those in the art and may be functional in, but are notlimited to, a bacterium, yeast, plant, or animal cell. The expressionvector can also include a translation regulatory sequence (e.g., anuntranslated 5′ sequence, an untranslated 3′ sequence, a poly A additionsite, or an internal ribosome entry site), a splicing sequence orsplicing regulatory sequence, and a transcription termination sequence.The vector can be capable of autonomous replication or it can integrateinto a host DNA.

In an alternative embodiment, the DNA molecule is fused to a reportergene such as β-glucuronidase gene, chloramphenicol-acetyltransferasegene, a gene encoding green fluorescent protein (and variants thereof),or red fluorescent protein firefly luciferase gene, among others. TheDNA molecule can also be fused to a nucleic acid encoding a polypeptideaffinity tag, e.g., glutathione S-transferase (GST), maltose E bindingprotein, or protein A, FLAG tag, hexa-histidine, or the influenza HAtag. The affinity tag or reporter fusion joins the reading frames of SEQID NO:1 or 2 to the reading frame of the reporter gene or the nucleicacid encoding the affinity tag such that a translational fusion isgenerated. Expression of the fusion gene results in translations of asingle polypeptide that includes both a nematode MDH-like region andreporter protein or the affinity tag. The fusion can also join afragment of the reading frame of SEQ ID NO:1 or 2. The fragment canencode a functional region of the MDH-like polypeptides, astructurally-intact domain, or an epitope (e.g., a peptide of about 8,10, 20, or 30 or more amino acids). A nematode MDH-like nucleic acidthat includes at least one of a regulatory region (e.g., a 5′ regulatoryregion, a promoter, an enhancer, a 5′ untranslated region, atranslational start site, a 3′ untranslated region, a polyadenylationsite, or a 3′ regulatory region) can also be fused to a heterologousnucleic acid. For example, the promoter of an MDH-like nucleic acid canbe fused to a heterologous nucleic acid, e.g., a nucleic acid encoding areporter protein.

Suitable cells to transform include any cell that can be transformedwith a nucleic acid molecule of the present invention. A transformedcell of the present invention is also herein referred to as arecombinant cell. Suitable cells can either be untransformed cells orcells that have already been transformed with at least one nucleic acidmolecule. Suitable cells for transformation according to the presentinvention can either be: (i) endogenously capable of expressing theMDH-like proteins or; (ii) capable of producing such proteins aftertransformation with at least one nucleic acid molecule of the presentinvention.

In an exemplary embodiment, a nucleic acid of the invention is used togenerate a transgenic nematode strain, e.g., a transgenic C. elegansstrain. To generate such a strain, nucleic acid is injected into thegonad of a nematode, thus generating a heritable extrachromosomal arraycontaining the nucleic acid (see, e.g., Mello et al. (1991) EMBO J.10:3959-3970). The transgenic nematode can be propagated to generate astrain harboring the transgene. Nematodes of the strain can be used inscreens to identify inhibitors specific for a M. incognita MDH-likegene.

Oligonucleotides

Also provided are oligonucleotides that can form stable hybrids with anucleic acid molecule of the present invention. The oligonucleotides canbe about 10 to 200 nucleotides, about 15 to 120 nucleotides, or about 17to 80 nucleotides in length, e.g., about 10, 20, 30, 40, 50, 60, 80,100, 120 nucleotides in length. The oligonucleotides can be used asprobes to identify nucleic acid molecules, primers to produce nucleicacid molecules, or therapeutic reagents to inhibit nematode MDH-likeprotein activity or production (e.g., antisense, triplex formation,ribozyme, and/or RNA drug-based reagents). The present inventionincludes oligonucleotides of RNA (single stranded (ss) RNA and doublestranded (ds) RNA), DNA, or derivatives of either. The invention extendsto the use of such oligonucleotides to protect non-nematode organisms(for example, plants and animals) from disease, e.g., using a technologydescribed herein. Appropriate oligonucleotide-containing therapeuticcompositions can be administered to a non-nematode organism usingtechniques known to those skilled in the art, including, but not limitedto, transgenic expression in plants or animals.

Primer sequences can be used to amplify an MDH-like nucleic acid orfragment thereof. For example, at least 10 cycles of PCR amplificationcan be used to obtain such an amplified nucleic acid. Primers can be atleast about 8-40, 10-30 or 14-25 nucleotides in length, and can annealto a nucleic acid “template molecule”, e.g. a template molecule encodingas a MDH-like genetic sequence, or a functional part thereof, or itscomplementary sequence. The nucleic acid primer molecule can be anynucleotide sequence of at least 10 nucleotides in length derived from,or contained within sequences depicted in SEQ ID NO:1 and/or SEQ IDNO:2, and their complements. The nucleic acid template molecule may bein a recombinant form, in a virus particle, bacteriophage particle,yeast cell, animal cell, plant cell, fungal cell, or bacterial cell. Aprimer can be chemically synthesized by routine methods.

This invention embodies any MDH-like sequences that are used to identifyand isolate similar genes from other organisms, including nematodes,prokaryotic organisms, and other eukaryotic organisms, such as otheranimals and/or plants.

In another embodiment, the invention provides oligonucleotides which arespecific for a M. incognita MDH-like nucleic acid molecule. Sucholigonucleotides can used in a PCR test to determine if an M. incognitanucleic acid is present in a sample, e.g., to monitor a disease causedby M. incognita.

Protein Production

Isolated MDH-like proteins from nematodes can be produced in a number ofways, including production and recovery of the recombinant proteinsand/or chemical synthesis of the protein. In one embodiment, an isolatednematode MDH-like protein is produced by culturing a cell, e.g., abacterial, fungal, plant, or animal cell, capable of expressing theprotein under conditions for effective production, and recovery of theprotein. The nucleic acid can be operably linked to a heterologouspromoter, e.g., an inducible promoter or a constitutive promoter.Effective growth conditions are typically, but are not necessarily, inliquid media comprising salts, water, carbon, nitrogen, phosphatesources, minerals, and other nutrients, but may be any solution in whichMDH-like proteins may be produced.

In one embodiment, recovery of the protein may refer to collecting thegrowth solution and need not involve additional steps of purification.Proteins of the present invention, however, can be purified usingstandard purification techniques, such as, but not limited to, affinitychromatography, thermaprecipitation, immunoaffinity chromatography,ammonium sulfate precipitation, ion exchange chromatography, filtration,electrophoresis, hydrophobic interaction chromatography, and others.

The MDH-like polypeptide can be fused to an affinity tag, e.g., apurification handle (e.g., glutathione-S-reductase, hexa-histidine,maltose binding protein, dihydrofolate reductases, or chitin bindingprotein) or an epitope tag (e.g., c-myc epitope tag, FLAG™ tag, orinfluenza HA tag). Affinity tagged and epitope tagged proteins can bepurified using routine art-known methods.

Antibodies Against MDH-like Polypeptides

Recombinant MDH-like gene products or derivatives thereof can be used toproduce immunologically interactive molecules, such as antibodies, orfunctional derivatives thereof. Useful antibodies include those thatbind to a polypeptide that has substantially the same sequence as theamino acid sequences recited in SEQ ID NO:3 and SEQ ID NO:4, or that hasat least 70% similarity over 50 or more amino acids to these sequences.In a preferred embodiment, the antibody specifically binds to apolypeptide having the amino acid sequence recited in SEQ ID NO:3 or 4.The antibodies can be antibody fragments and genetically engineeredantibodies, including single chain antibodies or chimeric antibodiesthat can bind to more than one epitope. Such antibodies may bepolyclonal or monoclonal and may be selected from naturally occurringantibodies or may be specifically raised to a recombinant MDH-likeprotein.

Antibodies can be derived by immunization with a recombinant or purifiedMDH-like gene or gene product. As used herein, the term “antibody”refers to an immunoglobulin, or fragment thereof. Examples of antibodyfragments include F(ab) and F(ab′)2 fragments, particularly functionalones able to bind epitopes. Such fragments can be generated byproteolytic cleavage, e.g., with pepsin, or by genetic engineering.Antibodies can be polyclonal, monoclonal, or recombinant. In addition,antibodies can be modified to be chimeric, or humanized. Further, anantibody can be coupled to a label or a toxin.

Antibodies can be generated against a full-length MDH-like protein, or afragment thereof, e.g., an antigenic peptide. Such polypeptides can becoupled to an adjuvant to improve immunogenicity. Polyclonal serum isproduced by injection of the antigen into a laboratory animal such as arabbit and subsequent collection of sera. Alternatively, the antigen isused to immunize mice. Lymphocytic cells are obtained from the mice andfused with myelomas to form hybridomas producing antibodies.

Peptides for generating MDH-Like antibodies can be about 8, 10, 15, 20,30 or more amino acid residues in length, e.g., a peptide of such lengthobtained from SEQ ID NO:3 or SEQ ID NO:4. Peptides or epitopes can alsobe selected from regions exposed on the surface of the protein, e.g.,hydrophilic or amphipathic regions. An epitope in the vicinity of theactive site can be selected such that an antibody binding such anepitope would block access to the active site. Antibodies reactive with,or specific for, any of these regions, or other regions or domainsdescribed herein are provided. An antibody to an MDH-like protein canmodulate an MDH-like activity.

Monoclonal antibodies, which can be produced by routine methods, areobtained in abundance and in homogenous form from hybridomas formed fromthe fusion of immortal cell lines (e.g., myelomas) with lymphocytesimmunized with MDH-like polypeptides such as those set forth in SEQ IDNO:3 and SEQ ID NO:4.

In addition, antibodies can be engineered, e.g., to produce a singlechain antibody (see, for example, Colcher, D. et al. (1999) Ann N Y AcadSci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). Instill another implementation, antibodies are selected or modified basedon screening procedures, e.g., by screening antibodies or fragmentsthereof from a phage display library.

Antibodies of the present invention have a variety of important useswithin the scope of this invention. For example, such antibodies can beused: (i) as therapeutic compounds to passively immunize an animal inorder to protect the animal from nematodes susceptible to antibodytreatment; (ii) as reagents in experimental assays to detect presence ofnematodes; (iii) as tools to screen for expression of the gene productin nematodes, animals, fungi, bacteria, and plants; and/or (iv) as apurification tool of MDH-like protein; (v) as MDH inhibitors/activatorsthat can be expressed or introduced into plants or animals fortherapeutic purposes.

An antibody against an MDH-like protein can be produce in a plant cell,e.g., in a transgenic plant or in culture (see, e.g., U.S. Pat. No.6,080,560).

Antibodies that specifically recognize M. incognita MDH-like proteinscan be used to identify an M. incognita nematode, and, thus, can be usedto monitor a disease caused by M. incognita.

Nucleic Acids Agents

Also featured are isolated nucleic acids which are antisense to nucleicacids encoding nematode MDH-like proteins. An “antisense” nucleic acidincludes a sequence that is complementary to the coding strand of anucleic acid encoding an MDH-like protein. The complementarity can be ina coding region of the coding strand or in a noncoding region, e.g., a5′ or 3′ untranslated region, e.g., the translation start site. Theantisense nucleic acid can be produced from a cellular promoter (e.g., aRNA polymerase II or III promoter), or can be introduced into a cell,e.g., using a liposome. For example, the antisense nucleic acid can be asynthetic oligonucleotide having a length of about 10, 15, 20, 30, 40,50, 75, 90, 120 or more nucleotides in length.

An antisense nucleic acid can be synthesized chemically or producedusing enzymatic reagents, e.g., a ligase. An antisense nucleic acid canalso incorporate modified nucleotides, and artificial backbonestructures, e.g., pbosphorothioate derivative, and acridine substitutednucleotides.

Ribozymes. The antisense nucleic acid can be a ribozyme. The ribozymecan be designed to specifically cleave RNA, e.g., an MDH-like MRNA.Methods for designing such ribozymes are described in U.S. Pat. No.5,093,246 or Haselhoff and Gerlach (1988) Nature 334:585-591. Forexample, the ribozyme can be a derivative of Tetrahymena L-19 IVS RNA inwhich the nucleotide sequence of the active site is modified to becomplementary to an MDH-like nucleic acid (see, e.g., Cech et al. U.S.Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742).

Peptide Nucleic acid (PNA). An antisense agent directed against anMDH-like nucleic acid can be a peptide nucleic acid (PNA). See Hyrup B.et al. (1996) Bioorganic & Medicinal Chemistry 4: 5-23) for methods anda description of the replacement of the deoxyribose phosphate backbonefor a pseudopeptide backbone. A PNA can specifically hybridize to DNAand RNA under conditions of low ionic strength as a result of itselectrostatic properties. The synthesis of PNA oligomers can beperformed using standard solid phase peptide synthesis protocols asdescribed in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc.Natl. Acad Sci. 93: 14670-675.

RNA Mediated Interference (RNAi). A double stranded RNA (dsRNA) moleculecan be used to inactivate a MDH-like gene in a cell by a process knownas RNA mediated-interference (RNAi; e.g., Fire etal. (1998) Nature391:806-811, and Gönczy et al. (2000) Nature 408:331-336). The dsRNAmolecule can have the nucleotide sequence of an MDH-like nucleic aciddescribed herein or a fragment thereof. The molecule can be injectedinto a cell, or a syncitia, e.g., a nematode gonad as described in Fireet al., supra.

Screening Assays

Another embodiment of the present invention is a method of identifying acompound capable of altering (e.g., inhibiting or enhancing) theactivity of MDH-like molecules. This method, also referred to as a“screening assay,” herein, includes, but is not limited to, thefollowing procedure: (i) contacting an isolated MDH-like protein with atest compound, under conditions in which, in the absence of the testcompound, the protein has MDH-like activity; and (ii) determining if thetest compound alters an MDH-like activity. Suitable inhibitors oractivators that alter a nematode MDH-like activity include compoundsthat interact directly with a nematode MDH-like protein, perhaps but notnecessarily, in the active. site. They can also interact with otherregions of the nematode MDH protein by binding to regions outside of theactive site, for example, by allosteric interaction.

Compounds. A test compound can be a large or small molecule, forexample, an organic compound with a molecular weight of about 100 to10,000; 200 to 5,000; 200 to 2000; or 200 to 1,000 daltons. A testcompound can be any chemical compound, for example, a small organicmolecule, a carbohydrate, a lipid, an amino acid, a polypeptide, anucleoside, a nucleic acid, or a peptide nucleic acid. Small moleculesinclude, but are not limited to, metabolites, metabolic analogues,peptides, peptidomimetics (e.g., peptoids), amino acids, amino acidanalogs, polynucleotides, polynucleotide analogs, nucleotides,nucleotide analogs, organic or inorganic compounds (e.g., includingheteroorganic and organometallic compounds). A metabolite or metabolicanalog can be malate and/or oxaloacetate, and derivatives thereofCompounds and components for synthesis of compounds can be obtained froma commercial chemical supplier, e.g., Sigma-Aldrich Corp. (St. Louis,Mo.). The test compound or compounds can be naturally occurring,synthetic, or both. A test compound can be the only substance assayed bythe method described herein. Alternatively, a collection of testcompounds can be assayed either consecutively or concurrently by themethods described herein.

Examples of known inhibitors of MDH proteins present in other organismsinclude amocarzine and/or suramin (Loiseau et a. (1993) Parasitol. Res.79:397-401). In addition, derivatives and mimetics of malate andoxaloacetate can be screened and/or used.

A high-throughput method can be used to screen large libraries ofchemicals. Such libraries of candidate compounds can be generated orpurchased, e.g., from Chembridge Corp. (San Diego, Calif.). Librariescan be designed to cover a diverse range of compounds. For example, alibrary can include 10,000, 50,000, or 100,000 or more unique compounds.Merely by way of illustration, a library can be constructed fromheterocycles including pyridines, indoles, quinolines, furans,pyrimidines, triazines, pyrroles, imidazoles, naphthalenes,benzimidazoles, piperidines, pyrazoles, benzoxazoles, pyrrolidines,thiphenes, thiazoles, benzothiazoles, and morpholines. Alternatively, aclass or category of compounds can be selected to mimic the chemicalstructures of malate, oxaloacetate, amocarzine and suramin. A librarycan be designed and synthesized to cover such classes of chemicals,e.g., as described in DeWitt et al., (1993) Proc. Natl. Acad Sci. USA.90:6909; Erb et al., (1994) Proc. Natl. Acad. Sci. USA91:11422;Zuckermann et al., (1994). J. Med. Chem. 37:2678; Cho et al.,(1993) Science 261:1303; Carrell et al., (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al., (1994) Angew. Chem. Int. Ed. Engl.33:2061; and in Gallop et al., (1994) J. Med. Chem. 37:1233.

Organism-based Assays. Organisms can be grown in small microtiterplates, e.g., 6-well, 32-well, 64-well, 96-well, 384-well plates.

In one embodiment, the organism is a nematode. The nematodes can begenetically modified. Non-limiting examples of such modified nematodesinclude: 1) M. incognita wild-type strains; 2) nematodes or nematodecells (e.g., C. elegans or M. incognita) having one or more MDH-likegenes inactivated (e.g., using RNA mediated interference); 3) nematodesor nematode cells expressing a heterologous MDH-like gene, e.g., anMDH-like gene from another species; and 4) nematodes or nematode cellshaving one or more endogenous MDH-like genes inactivated and expressinga heterologous MDH-like gene, e.g., a M. incognita MDH-like gene asdescribed herein.

A plurality of candidate compounds, e.g., a combinatorial library, isscreened. The library can be provided in a format that is amenable forrobotic manipulation, e.g., in microtitre plates. Compounds can be addedto the wells of the microtiter plates. Following compound addition andincubation, viability and/or reproductive properties of the nematodes ornematode cells are monitored.

The compounds can also be pooled, and the pools tested. Positive poolsare split for subsequent analysis. Regardless of the method, compoundsthat decrease the viability or reproductive ability of nematodes,nematode cells, or progeny of the nematodes are considered leadcompounds.

In another embodiment, the organism is a microorganism, e.g., a yeast orbacterium.

For example, an E, coli strain having a deletion or inactivatingmutation in an E, coli MDH-like gene, but expressing a nematode MDH-likegene can be used. The generation of such strains is routine in the art.As described above for nematodes and nematode cells, the microorganismcan be grown in microtitre plates, each well having a differentcandidate compound or pool of candidate compounds. Growth is monitoredduring or after the assay to determine if the compound or pool ofcompounds is a modulator of a nematode MDH-like polypeptide.

In Vitro Activity Assays. The screening assay can be an in vitroactivity assay. For example, a nematode MDH-like polypeptide is purifiedas described above. The polypeptide is disposed in an assay container,e.g., a well of microtitre plate. A candidate compound is added to theassay container, and the MDH-like activity is measured. Optionally, theactivity is compared to the activity measured in a control container inwhich no candidate compound is disposed or in which an inert ornon-functional compound is disposed.

An MDH-like activity assay can be an assay for the conversion of malateto oxaloacetate or for the conversion of oxaloacetate to malate.

In one method for measuring the conversion of malate to oxaloacetate,the MDH-like polypeptide is disposed in a reaction mixture of 50 mMTris-HCl pH 8.0, 0.2 mM NAD, and 6.7 mM malate (see, e.g., Prichard andSchofield (1968) Comp. Biochem. Physiol. 25:1005-19). The addition ofmalate to the reaction mixture is used to initiate the reaction. Thereaction can be monitored by following the increasing absorbance at 340nm, e.g., in a spectrophotometer. An increase in absorbance at 340 nm islinearly proportional to the amount of NADH formed. The kinetic andequilibrium parameters of the reaction are determined, e.g., usingart-known methods such as Lineweaver-Burk plots and Dixon plots. Theassay can be used to measure inhibition coefficients, e.g., a K₁, of acandidate compound, by measuring reaction rates at varyingconcentrations of the candidate compound.

In one method for measuring the conversion of oxaloacetate to malate,the MDH-like polypeptide is disposed in a reaction mixture of 50 nMTris-HCl pH 8.0, 0.2 mM NADH₂, and 0.33 mM oxaloacetate (see, e.g.,Prichard and Schofield, supra). The addition of oxaloacetate to thereaction mixture is used to initiate the reaction. The reaction ismonitored by following the decreasing absorbance at 340 nm, e.g., in aspectrophotometer. In another example, the reaction is monitored in amixture of 54 mM triethanolamine pH 7.6, 5 mM EDTA, 0.17 mM NADH₂, and0.33 mM oxaloacetate (see, e.g., Shonk and Boxer (1964) Cancer Res.24:709-721). These assays can be used to measure the ability of acandidate compound to inhibit the conversion of oxaloacatetate to malateby a nematode MDH-like polypeptide.

In Vitro Binding Assays. The screening assay can also be a cell-freebinding assay, e.g., an assay to identify compounds that bind a nematodeMDH-like polypeptide. For example, a nematode MDH-like polypeptide canbe purified and labeled. The labeled polypeptide is contacted to beads,each bead have a tag detectable by mass spectroscopy, and test compound,e.g., a compound synthesized by combinatorial chemical methods. Beads towhich the labeled polypeptide is bound are identified and analyzed bymass spectroscopy. The beads can be generated using “split-and-pool”synthesis. The method can further include a second assay (e.g., the MDHactivity assay described above) to determine if the compound alters theactivity of the MDH-like polypeptide.

Optimization of a Compound. Once a lead compound has been identified,standard principles of medicinal chemistry can be used to producederivatives of the compound. Derivatives can be screened for improvedpharmacological properties, for example, efficacy, pharmacokinetics,stability, solubility, and clearance. The moieties responsible for acompound's activity in the above-described assays can be delineated byexamination of structure-activity relationships (SAR) as is commonlypracticed in the art. A person of ordinary skill in chemistry couldmodify moieties on a lead compound and measure the effects of themodification on the efficacy of the compound to thereby producederivatives with increased potency. For an example, see Nagarajan et al.(1988) J. Antibiot. 41:1430-8. A modification can include N-acylation,amination, amidation, oxidation, reduction, alkylation, esterification,and hydroxylation. Furthermore, if the biochemical target of the leadcompound is known or determined, the structure of the target and thelead compound can inform the design and optimization of derivatives.Molecular modeling software is commercially available (e.g., fromMolecular Simulations, Inc.). “SAR by NMR,” as described in Shuker etal. (1996) Science 274:1531-4, can be used to design ligands withincreased affinity, e.g., by joining lower-affinity ligands.

A preferred compound is one that inhibits an MDH-like polypeptide andthat is not substantially toxic to plants, animals, or humans. By “notsubstantially toxic” it is meant that the compound does notsubstantially affect the respective plant, animal, or human MDHproteins. Thus, particularly desirable inhibitors of M. incognita MDH1and MDH2 do not substantially inhibit MDH-like polypeptides of cotton,tobacco, pepper, and tomato. In addition, a preferred inhibitor inhibitsboth M. incognita MDH1 and MDH2.

Standard pharmaceutical procedures can be used to assess the toxicityand therapeutic efficacy of a modulator of an MDH-like activity. TheLD50 (the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population can be measured incell cultures, experimental plants (e.g., in laboratory or fieldstudies), or experimental animals. Optionally, a therapeutic index canbe determined which is expressed as the ratio: LD50/ED50. Hightherapeutic indices are indicative of a compound being an effectiveMDH-like inhibitor, while not causing undue toxicity or side-effects toa subject (e.g., a host plant or host animal).

Alternatively, the ability of a candidate compound to modulate anon-nematode MDH-like polypeptide is assayed, e.g., by a methoddescribed herein. For example, the inhibition constant of a candidatecompound for a mammalian MDH-like polypeptide or a plant MDH-likepolypeptide (e.g., an MDH-like polypeptide from cotton, tobacco, pepper,tomato; Malate Dehydrogenase (Tomato), GenBank® accession number T06402;GI:7431232. Malate Dehydrogenase (Tobacco), GenBank® accession numberCAB45387; GI:5123836) can be measured and compared to the inhibitionconstant for a nematode MDH-like polypeptide. (Sasser and Carter (1985)An Advanced Treatise on Meloidogyne, Vol. 1, North Carolina StateUniversity Graphics; Sasser (1980) Plant Disease 64, 36-41).

The aforementioned analyses can be used to identify and/or design amodulator with specificity for nematode MDH-like polypeptide over plantor other animal (e.g., mammalian) MDH-like polypeptides. Suitablenematodes to target are any nematodes with the MDH-like proteins orproteins that can be targeted by a compound that otherwise inhibits,reduces, activates, or generally effects the activity of nematode MDHproteins.

Inhibitors of nematode MDH-like proteins can also be used to identifyMDH-like proteins in the nematode or other organisms using proceduresknown in the art, such a affinity chromatography. For example, a knowninhibitor may be linked to a resin and a nematode extract passed overthe resin, allowing any MDH-like proteins that bind the inhibitor tobind the resin. Subsequent biochemical techniques familiar to thoseskilled in the art can be performed to purify and identify boundMDH-like proteins.

Agricultural Compositions

A compound that is identified as an MDH-like polypeptide inhibitor canbe formulated as a composition that is applied to plants in order toconfer nematode resistance. The composition can be prepared in asolution, e.g., an aqueous solution, at a concentration from about0.005% to 10%, or about 0.01% to 1%, or about 0.1% to 0.5% by weight.The solution can include an organic solvent, e.g., glycerol or ethanol.The composition can be formulated with one or more agriculturallyacceptable carriers. Agricultural carriers can include: clay, talc,bentonite, diatomaceous earth, kaolin, silica, benzene, xylene, toluene,kerosene, N-methylpyrrolidone, alcohols (methanol, ethanol, isopropanol,n-butanol, ethylene glycol, propylene glycol, and the like), and ketones(acetone, methylethyl ketone, cyclohexanone, and the like). Theformulation can optionally further include stabilizers, spreadingagents, wetting extenders, dispersing agents, sticking agents,disintegrators, and other additives, and can be prepared as a liquid, awater-soluble solid (e.g., tablet, powder or granule), or a paste.

Prior to application, the solution can be combined with another desiredcomposition such another antihelmintic agent, germicide, fertilizer,plant growth regulator and the like. The solution may be applied to theplant tissue, for example, by spraying, e.g., with an atomizer, bydrenching, by pasting, or by manual application, e.g., with a sponge.The solution can also be distributed from an airborne source, e.g., anaircraft or other aerial object, e.g., a fixture mounted with anapparatus for spraying the solution, the fixture being of sufficientheight to distribute the solution to the desired plant tissues.Alternatively, the composition can be applied to plant tissue from avolatile or airborne source. The source is placed in the vicinity of theplant tissue and the composition is dispersed by diffusion through theatmosphere. The source and the plant tissue to be contacted can beenclosed in an incubator, growth chamber, or greenhouse, or can be insufficient proximity that they can be outdoors.

If the composition is distributed systemically thorough the plant, thecomposition can be applied to tissues other than the leaves, e.g., tothe stems or roots. Thus, the composition can be distributed byirrigation. The composition can also be injected directly into roots orstems.

A skilled artisan would be able to determine an appropriate dosage forformulation of the active ingredient of the composition. For example,the ED50 can be determined as described above from experimental data.The data can be obtained by experimentally varying the dose of theactive ingredient to identify a dosage effective for killing a nematode,while not causing toxicity in the host plant Or host animal (i.e.non-nematode animal).

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

15 1 1327 DNA Meloidogyne incognita CDS (55)...(1152) misc_feature(1)...(1327) n = A,T,C or G 1 caagtttgag atatttaaat tattattttggtgctaagaa aaattttgtg aaaa atg 57 Met 1 aat tat tca aag gat gcc cca gaattt gtt gtg tct cca aaa gat gca 105 Asn Tyr Ser Lys Asp Ala Pro Glu PheVal Val Ser Pro Lys Asp Ala 5 10 15 cgc gaa ttt gtt gta aaa tgt atg caaaca gtt gga aca tcc cct gac 153 Arg Glu Phe Val Val Lys Cys Met Gln ThrVal Gly Thr Ser Pro Asp 20 25 30 cat gct ggt caa tta gca gat cta tta ttggat gct gat ctt gtt gga 201 His Ala Gly Gln Leu Ala Asp Leu Leu Leu AspAla Asp Leu Val Gly 35 40 45 cac tat agt cat ggt cta aat cga ctt cat atttat gtg gat gac gtc 249 His Tyr Ser His Gly Leu Asn Arg Leu His Ile TyrVal Asp Asp Val 50 55 60 65 aaa aac gga gtt aaa gga aat gga gtt cca aaagtg tta aaa caa aaa 297 Lys Asn Gly Val Lys Gly Asn Gly Val Pro Lys ValLeu Lys Gln Lys 70 75 80 gga ggc act gct tgg gtt gat gga gaa aat ctt ctgggt gca gtt gtt 345 Gly Gly Thr Ala Trp Val Asp Gly Glu Asn Leu Leu GlyAla Val Val 85 90 95 gga aac ttc tgt acc gac ttg gct att aaa ttg gct aaagaa ttt ggc 393 Gly Asn Phe Cys Thr Asp Leu Ala Ile Lys Leu Ala Lys GluPhe Gly 100 105 110 gtt gct tgg gtg gta aca aaa aat tct aat cat tat ggagct tgt caa 441 Val Ala Trp Val Val Thr Lys Asn Ser Asn His Tyr Gly AlaCys Gln 115 120 125 cat tat act aag aaa att gca aat gca gga atg gtg ggaatg tct ttt 489 His Tyr Thr Lys Lys Ile Ala Asn Ala Gly Met Val Gly MetSer Phe 130 135 140 145 aca aat aca tcg cct ctc atg ttc ccc tgc cga tcttct gag att gga 537 Thr Asn Thr Ser Pro Leu Met Phe Pro Cys Arg Ser SerGlu Ile Gly 150 155 160 ctt ggt aca aac cct ctt tct tgt tgt gtc aac tcggaa aag aca gga 585 Leu Gly Thr Asn Pro Leu Ser Cys Cys Val Asn Ser GluLys Thr Gly 165 170 175 gac agt ttt ttg tta gac atg gct acg aca act gttgct ctt gga aag 633 Asp Ser Phe Leu Leu Asp Met Ala Thr Thr Thr Val AlaLeu Gly Lys 180 185 190 gta gag ctg gca gat tgt cgc ggt aaa aca caa attccc tcc aca tgg 681 Val Glu Leu Ala Asp Cys Arg Gly Lys Thr Gln Ile ProSer Thr Trp 195 200 205 ggt gcc gat tct aaa ggc aat cca tcg act gat acacaa gtt gtt tta 729 Gly Ala Asp Ser Lys Gly Asn Pro Ser Thr Asp Thr GlnVal Val Leu 210 215 220 225 cac ggt ggc gga ctt ttg cct tta ggc ggt atagaa gag acg gga tct 777 His Gly Gly Gly Leu Leu Pro Leu Gly Gly Ile GluGlu Thr Gly Ser 230 235 240 tac aaa gga acg ggt ctt tca atg atg ggt gaattg ttt tgt gga att 825 Tyr Lys Gly Thr Gly Leu Ser Met Met Gly Glu LeuPhe Cys Gly Ile 245 250 255 ttg gca ggg tca agt ttt gga aaa aat gta cgatta tgg ggg caa tca 873 Leu Ala Gly Ser Ser Phe Gly Lys Asn Val Arg LeuTrp Gly Gln Ser 260 265 270 cac aaa gcc gct gac aat ggc caa tgt ttt gttgct att gat caa gaa 921 His Lys Ala Ala Asp Asn Gly Gln Cys Phe Val AlaIle Asp Gln Glu 275 280 285 tgt ttt gcc cca gga ttt gct cct cgt tta caacaa ttt ttg gat gaa 969 Cys Phe Ala Pro Gly Phe Ala Pro Arg Leu Gln GlnPhe Leu Asp Glu 290 295 300 305 aca cgg aat ttg aaa ccg att tct gaa gaaaag cct gtt cta gtg cct 1017 Thr Arg Asn Leu Lys Pro Ile Ser Glu Glu LysPro Val Leu Val Pro 310 315 320 gga gat cct gaa aga atg aat aca gaa tatagc caa aag gct gga ggt 1065 Gly Asp Pro Glu Arg Met Asn Thr Glu Tyr SerGln Lys Ala Gly Gly 325 330 335 ttg gta tac caa gaa ggg cag ata aaa gctttg gaa gag ttg gcc aca 1113 Leu Val Tyr Gln Glu Gly Gln Ile Lys Ala LeuGlu Glu Leu Ala Thr 340 345 350 aaa tgt gat gtt caa atg ttc tca tac aaacga cta aaa tgaggatgag 1162 Lys Cys Asp Val Gln Met Phe Ser Tyr Lys ArgLeu Lys 355 360 365 atttaaatat ttttttgtgt agctgaaact gacttcaaacgagaaatgaa caatttccta 1222 aaaagcagtt agataagggt ttatttttca tttatttattttttaacctc attttttata 1282 tacgaataaa attaatgctc naaaaaaaaa aaaaaaaaaaaaaaa 1327 2 2088 DNA Meloidogyne incognita CDS (28)...(1125)misc_feature (1)...(2088) n = A,T,C or G 2 tggtgctaag aaaaattttg tgcgaaaatg aat tat tca aag gat gcc cca gaa 54 Met Asn Tyr Ser Lys Asp Ala ProGlu 1 5 ttt gtt gtc tct cca aaa gat gct cgc gaa ttt gtt gta aaa tgt atg102 Phe Val Val Ser Pro Lys Asp Ala Arg Glu Phe Val Val Lys Cys Met 1015 20 25 caa aca gtt gga aca tcc cct gac cat gct ggt caa tta gca gat ctc150 Gln Thr Val Gly Thr Ser Pro Asp His Ala Gly Gln Leu Ala Asp Leu 3035 40 tta tta gat gct gat ctt gtt ggg cat tac agt cat ggt cta aat cgg198 Leu Leu Asp Ala Asp Leu Val Gly His Tyr Ser His Gly Leu Asn Arg 4550 55 ctt cat att tat gtg gat gac gtc aaa aat gga gtt aaa gga aat gga246 Leu His Ile Tyr Val Asp Asp Val Lys Asn Gly Val Lys Gly Asn Gly 6065 70 gtt cca aaa gtg tta aaa caa aaa gga ggc act gct tgg gtg gat gga294 Val Pro Lys Val Leu Lys Gln Lys Gly Gly Thr Ala Trp Val Asp Gly 7580 85 gaa aat ctt ttg ggt gca gtt gtt ggc aac ttc tgt acc gat ttg gct342 Glu Asn Leu Leu Gly Ala Val Val Gly Asn Phe Cys Thr Asp Leu Ala 9095 100 105 att aaa ttg gct aaa gaa ttt ggt gtt gct tgg gtg gta aca aaaaat 390 Ile Lys Leu Ala Lys Glu Phe Gly Val Ala Trp Val Val Thr Lys Asn110 115 120 tct aat cat tat gga gct ngt caa cat tat act aag aaa att gcgaat 438 Ser Asn His Tyr Gly Ala Xaa Gln His Tyr Thr Lys Lys Ile Ala Asn125 130 135 gca gga atg gtg gga atg tca ttt aca aat act tca cct ctc atgttc 486 Ala Gly Met Val Gly Met Ser Phe Thr Asn Thr Ser Pro Leu Met Phe140 145 150 ccc tgc cgt tct tct gag atc gga cta ggc aca aac cct ctt tcttgt 534 Pro Cys Arg Ser Ser Glu Ile Gly Leu Gly Thr Asn Pro Leu Ser Cys155 160 165 tgt gcc aac tcg gaa aag aca gaa gac agt ttt ttg tta gac atggct 582 Cys Ala Asn Ser Glu Lys Thr Glu Asp Ser Phe Leu Leu Asp Met Ala170 175 180 185 act aca act gtt gct cta gga aag gtt gag ctg gca aat tgtcgc ggt 630 Thr Thr Thr Val Ala Leu Gly Lys Val Glu Leu Ala Asn Cys ArgGly 190 195 200 aaa aca caa att ccc tca gca tgg ggt gcc gat tct aaa ggcaat cca 678 Lys Thr Gln Ile Pro Ser Ala Trp Gly Ala Asp Ser Lys Gly AsnPro 205 210 215 tca aca gac aca caa gtt gtt tta cat ggt ggc gga ctt ttgcct tta 726 Ser Thr Asp Thr Gln Val Val Leu His Gly Gly Gly Leu Leu ProLeu 220 225 230 ggc ggt ata gaa gag acg gga tct tac aaa gga acg ggt ctctca atg 774 Gly Gly Ile Glu Glu Thr Gly Ser Tyr Lys Gly Thr Gly Leu SerMet 235 240 245 atg ggt gaa ttg ttt tgt gga att ttg gca ggg tca agt tttgga aaa 822 Met Gly Glu Leu Phe Cys Gly Ile Leu Ala Gly Ser Ser Phe GlyLys 250 255 260 265 aat gta cga tta tgg ggg caa tca cac aaa gcc gct gacaat ggc caa 870 Asn Val Arg Leu Trp Gly Gln Ser His Lys Ala Ala Asp AsnGly Gln 270 275 280 tgt ttt gtt gct att gat caa gaa tgt ttt gcc cca ggattt gct cct 918 Cys Phe Val Ala Ile Asp Gln Glu Cys Phe Ala Pro Gly PheAla Pro 285 290 295 cgt tta caa caa ttt ttg gat gaa aca cgg aat ttg aaaccg att tct 966 Arg Leu Gln Gln Phe Leu Asp Glu Thr Arg Asn Leu Lys ProIle Ser 300 305 310 gaa gaa aag cct gtt cta gtg cct gga gat cct gaa agaatg aat aca 1014 Glu Glu Lys Pro Val Leu Val Pro Gly Asp Pro Glu Arg MetAsn Thr 315 320 325 gaa tat agc caa aag gct gga ggt ttg gta tac caa gaaggg cag ata 1062 Glu Tyr Ser Gln Lys Ala Gly Gly Leu Val Tyr Gln Glu GlyGln Ile 330 335 340 345 aaa gct ttg gaa gag ttg gcc aca aaa tgt gat gttcaa atg ttc tca 1110 Lys Ala Leu Glu Glu Leu Ala Thr Lys Cys Asp Val GlnMet Phe Ser 350 355 360 tac aaa cga cta aaa tgaggatgag atttaaatatttttttgtgt agctgaaact 1165 Tyr Lys Arg Leu Lys 365 gacttcaaac gagaaatgaacaatttccta aaaagcagtt agataagggt ttatttttca 1225 tttatttatt ttttaacctcattttttata tacgaagcag atatgactga aactggaggt 1285 ggtgattctg ttgaatctgcaagtgtttat gctaactctg tttgtgaaat gtgcggaaat 1345 tatgaggttc aacttcaaacaattcaaagc agtcaggata ctctcaggga gaaattggca 1405 gctgctaaag aattgtatgagaaatatggc aaggaattga cagaagagag gcattatcga 1465 aaggaattgg aaattaaatttgctgcttta aatgaagaaa ctgaagggaa aattcagcaa 1525 tgtattacca atacagaagactttgacagc gtattgcctt ctcagtaaaa aacaanaagc 1585 tgatttgtct gttttggaatcncaattaga attggctagg aatcgtcaaa aagagcttca 1645 agaacaattg gttttgttaaatgaaaggta tgaaaaactt ttacatttaa aatctcaatg 1705 tgctgaagaa atgcgtgaacaacaaattga actgcctcaa acagttgaag aacttcaatt 1765 tttggcattg cagttganagaggaattgat aactgaacgt gcagcacgtg agcatgaaag 1825 gagggaatta aatgatgaattggctatggc acgtcaacag cttgttgaat tggaaatttg 1885 tccnagagaa aatgaagaatgaattttatg atatataaaa atatatttat tttgctcaaa 1945 tagnttttat aaattttaagagctgataga aaaatttagt tttgnaattt ttgaagaata 2005 tattttntac ggtttgcacnccttagaatg gttttgtttt aataaatgcn cnggttggna 2065 aaaaaaaaaa aaaaaaaaaaaaa 2088 3 366 PRT Meloidogyne incognita 3 Met Asn Tyr Ser Lys Asp AlaPro Glu Phe Val Val Ser Pro Lys Asp 1 5 10 15 Ala Arg Glu Phe Val ValLys Cys Met Gln Thr Val Gly Thr Ser Pro 20 25 30 Asp His Ala Gly Gln LeuAla Asp Leu Leu Leu Asp Ala Asp Leu Val 35 40 45 Gly His Tyr Ser His GlyLeu Asn Arg Leu His Ile Tyr Val Asp Asp 50 55 60 Val Lys Asn Gly Val LysGly Asn Gly Val Pro Lys Val Leu Lys Gln 65 70 75 80 Lys Gly Gly Thr AlaTrp Val Asp Gly Glu Asn Leu Leu Gly Ala Val 85 90 95 Val Gly Asn Phe CysThr Asp Leu Ala Ile Lys Leu Ala Lys Glu Phe 100 105 110 Gly Val Ala TrpVal Val Thr Lys Asn Ser Asn His Tyr Gly Ala Cys 115 120 125 Gln His TyrThr Lys Lys Ile Ala Asn Ala Gly Met Val Gly Met Ser 130 135 140 Phe ThrAsn Thr Ser Pro Leu Met Phe Pro Cys Arg Ser Ser Glu Ile 145 150 155 160Gly Leu Gly Thr Asn Pro Leu Ser Cys Cys Val Asn Ser Glu Lys Thr 165 170175 Gly Asp Ser Phe Leu Leu Asp Met Ala Thr Thr Thr Val Ala Leu Gly 180185 190 Lys Val Glu Leu Ala Asp Cys Arg Gly Lys Thr Gln Ile Pro Ser Thr195 200 205 Trp Gly Ala Asp Ser Lys Gly Asn Pro Ser Thr Asp Thr Gln ValVal 210 215 220 Leu His Gly Gly Gly Leu Leu Pro Leu Gly Gly Ile Glu GluThr Gly 225 230 235 240 Ser Tyr Lys Gly Thr Gly Leu Ser Met Met Gly GluLeu Phe Cys Gly 245 250 255 Ile Leu Ala Gly Ser Ser Phe Gly Lys Asn ValArg Leu Trp Gly Gln 260 265 270 Ser His Lys Ala Ala Asp Asn Gly Gln CysPhe Val Ala Ile Asp Gln 275 280 285 Glu Cys Phe Ala Pro Gly Phe Ala ProArg Leu Gln Gln Phe Leu Asp 290 295 300 Glu Thr Arg Asn Leu Lys Pro IleSer Glu Glu Lys Pro Val Leu Val 305 310 315 320 Pro Gly Asp Pro Glu ArgMet Asn Thr Glu Tyr Ser Gln Lys Ala Gly 325 330 335 Gly Leu Val Tyr GlnGlu Gly Gln Ile Lys Ala Leu Glu Glu Leu Ala 340 345 350 Thr Lys Cys AspVal Gln Met Phe Ser Tyr Lys Arg Leu Lys 355 360 365 4 366 PRTMeloidogyne incognita VARIANT (1)...(366) Xaa = Any Amino Acid 4 Met AsnTyr Ser Lys Asp Ala Pro Glu Phe Val Val Ser Pro Lys Asp 1 5 10 15 AlaArg Glu Phe Val Val Lys Cys Met Gln Thr Val Gly Thr Ser Pro 20 25 30 AspHis Ala Gly Gln Leu Ala Asp Leu Leu Leu Asp Ala Asp Leu Val 35 40 45 GlyHis Tyr Ser His Gly Leu Asn Arg Leu His Ile Tyr Val Asp Asp 50 55 60 ValLys Asn Gly Val Lys Gly Asn Gly Val Pro Lys Val Leu Lys Gln 65 70 75 80Lys Gly Gly Thr Ala Trp Val Asp Gly Glu Asn Leu Leu Gly Ala Val 85 90 95Val Gly Asn Phe Cys Thr Asp Leu Ala Ile Lys Leu Ala Lys Glu Phe 100 105110 Gly Val Ala Trp Val Val Thr Lys Asn Ser Asn His Tyr Gly Ala Xaa 115120 125 Gln His Tyr Thr Lys Lys Ile Ala Asn Ala Gly Met Val Gly Met Ser130 135 140 Phe Thr Asn Thr Ser Pro Leu Met Phe Pro Cys Arg Ser Ser GluIle 145 150 155 160 Gly Leu Gly Thr Asn Pro Leu Ser Cys Cys Ala Asn SerGlu Lys Thr 165 170 175 Glu Asp Ser Phe Leu Leu Asp Met Ala Thr Thr ThrVal Ala Leu Gly 180 185 190 Lys Val Glu Leu Ala Asn Cys Arg Gly Lys ThrGln Ile Pro Ser Ala 195 200 205 Trp Gly Ala Asp Ser Lys Gly Asn Pro SerThr Asp Thr Gln Val Val 210 215 220 Leu His Gly Gly Gly Leu Leu Pro LeuGly Gly Ile Glu Glu Thr Gly 225 230 235 240 Ser Tyr Lys Gly Thr Gly LeuSer Met Met Gly Glu Leu Phe Cys Gly 245 250 255 Ile Leu Ala Gly Ser SerPhe Gly Lys Asn Val Arg Leu Trp Gly Gln 260 265 270 Ser His Lys Ala AlaAsp Asn Gly Gln Cys Phe Val Ala Ile Asp Gln 275 280 285 Glu Cys Phe AlaPro Gly Phe Ala Pro Arg Leu Gln Gln Phe Leu Asp 290 295 300 Glu Thr ArgAsn Leu Lys Pro Ile Ser Glu Glu Lys Pro Val Leu Val 305 310 315 320 ProGly Asp Pro Glu Arg Met Asn Thr Glu Tyr Ser Gln Lys Ala Gly 325 330 335Gly Leu Val Tyr Gln Glu Gly Gln Ile Lys Ala Leu Glu Glu Leu Ala 340 345350 Thr Lys Cys Asp Val Gln Met Phe Ser Tyr Lys Arg Leu Lys 355 360 3655 1098 DNA Meloidogyne incognita 5 atgaattatt caaaggatgc cccagaatttgttgtgtctc caaaagatgc acgcgaattt 60 gttgtaaaat gtatgcaaac agttggaacatcccctgacc atgctggtca attagcagat 120 ctattattgg atgctgatct tgttggacactatagtcatg gtctaaatcg acttcatatt 180 tatgtggatg acgtcaaaaa cggagttaaaggaaatggag ttccaaaagt gttaaaacaa 240 aaaggaggca ctgcttgggt tgatggagaaaatcttctgg gtgcagttgt tggaaacttc 300 tgtaccgact tggctattaa attggctaaagaatttggcg ttgcttgggt ggtaacaaaa 360 aattctaatc attatggagc ttgtcaacattatactaaga aaattgcaaa tgcaggaatg 420 gtgggaatgt cttttacaaa tacatcgcctctcatgttcc cctgccgatc ttctgagatt 480 ggacttggta caaaccctct ttcttgttgtgtcaactcgg aaaagacagg agacagtttt 540 ttgttagaca tggctacgac aactgttgctcttggaaagg tagagctggc agattgtcgc 600 ggtaaaacac aaattccctc cacatggggtgccgattcta aaggcaatcc atcgactgat 660 acacaagttg ttttacacgg tggcggacttttgcctttag gcggtataga agagacggga 720 tcttacaaag gaacgggtct ttcaatgatgggtgaattgt tttgtggaat tttggcaggg 780 tcaagttttg gaaaaaatgt acgattatgggggcaatcac acaaagccgc tgacaatggc 840 caatgttttg ttgctattga tcaagaatgttttgccccag gatttgctcc tcgtttacaa 900 caatttttgg atgaaacacg gaatttgaaaccgatttctg aagaaaagcc tgttctagtg 960 cctggagatc ctgaaagaat gaatacagaatatagccaaa aggctggagg tttggtatac 1020 caagaagggc agataaaagc tttggaagagttggccacaa aatgtgatgt tcaaatgttc 1080 tcatacaaac gactaaaa 1098 6 1098DNA Meloidogyne incognita misc_feature (1)...(1098) n = A,T,C or G 6atgaattatt caaaggatgc cccagaattt gttgtctctc caaaagatgc tcgcgaattt 60gttgtaaaat gtatgcaaac agttggaaca tcccctgacc atgctggtca attagcagat 120ctcttattag atgctgatct tgttgggcat tacagtcatg gtctaaatcg gcttcatatt 180tatgtggatg acgtcaaaaa tggagttaaa ggaaatggag ttccaaaagt gttaaaacaa 240aaaggaggca ctgcttgggt ggatggagaa aatcttttgg gtgcagttgt tggcaacttc 300tgtaccgatt tggctattaa attggctaaa gaatttggtg ttgcttgggt ggtaacaaaa 360aattctaatc attatggagc tngtcaacat tatactaaga aaattgcgaa tgcaggaatg 420gtgggaatgt catttacaaa tacttcacct ctcatgttcc cctgccgttc ttctgagatc 480ggactaggca caaaccctct ttcttgttgt gccaactcgg aaaagacaga agacagtttt 540ttgttagaca tggctactac aactgttgct ctaggaaagg ttgagctggc aaattgtcgc 600ggtaaaacac aaattccctc agcatggggt gccgattcta aaggcaatcc atcaacagac 660acacaagttg ttttacatgg tggcggactt ttgcctttag gcggtataga agagacggga 720tcttacaaag gaacgggtct ctcaatgatg ggtgaattgt tttgtggaat tttggcaggg 780tcaagttttg gaaaaaatgt acgattatgg gggcaatcac acaaagccgc tgacaatggc 840caatgttttg ttgctattga tcaagaatgt tttgccccag gatttgctcc tcgtttacaa 900caatttttgg atgaaacacg gaatttgaaa ccgatttctg aagaaaagcc tgttctagtg 960cctggagatc ctgaaagaat gaatacagaa tatagccaaa aggctggagg tttggtatac 1020caagaagggc agataaaagc tttggaagag ttggccacaa aatgtgatgt tcaaatgttc 1080tcatacaaac gactaaaa 1098 7 372 PRT Caenorhabidits elegans 7 Met Thr IleLys Asp Lys Arg Glu Phe Asn Glu Thr Asp Glu Ile Val 1 5 10 15 Ile SerLys Glu Lys Leu Asp Ser Phe Val Leu Glu Cys Leu Ala Lys 20 25 30 Ala GlyCys Thr Gly Asp His Ala Gln Gln Leu Ala Glu Thr Leu Leu 35 40 45 Cys SerAsp Tyr Arg Gly His Tyr Ser His Gly Ile Asn Arg Leu His 50 55 60 Ile TyrVal His Asp Leu Met Met Lys Ser Thr Ala Val Thr Gly Thr 65 70 75 80 ProGln Val Leu Lys Ser Lys Gly Ser Thr Ala Trp Val Asp Gly Asn 85 90 95 AsnLeu Leu Gly Pro Val Val Gly Asn Phe Cys Met Gln Leu Ala Val 100 105 110Glu Lys Ala Lys Glu Phe Gly Ile Gly Trp Val Val Cys Arg Asn Ser 115 120125 Asn His Phe Gly Ile Ala Gly Trp Tyr Ala Asp Phe Ala Cys Arg Asn 130135 140 Gly Leu Val Gly Met Ala Phe Thr Asn Thr Ser Pro Cys Val Phe Pro145 150 155 160 Thr Gly Ser Arg Glu Lys Ser Leu Gly Ser Asn Pro Ile CysMet Ala 165 170 175 Ala Pro Gly Met Glu Gly Asp Ser Phe Phe Leu Asp MetAla Ser Thr 180 185 190 Thr Val Ala Tyr Gly Lys Ile Glu Val Val Asp ArgLys Gly Glu Thr 195 200 205 Tyr Ile Pro Gly Ser Trp Gly Ala Asp Lys AsnGly Asp Glu Thr His 210 215 220 Asn Pro Lys Glu Val Leu Asp Gly Gly GlyLeu Gln Pro Leu Gly Gly 225 230 235 240 Ser Glu Ile Thr Gly Gly Tyr LysGly Thr Gly Leu Cys Met Met Val 245 250 255 Glu Val Leu Cys Gly Ile MetGly Gly Ser Ala Phe Gly Lys Asn Ile 260 265 270 Arg Gln Trp Gln Thr ThrSer Lys Thr Ala Asp Leu Gly Gln Cys Phe 275 280 285 Val Ala Ile Asp ProGlu Cys Phe Ala Pro Gly Phe Ser Asn Arg Leu 290 295 300 Gln Glu Phe CysAsp Glu Thr Arg Asn Leu Asn Pro Ile Asn Pro Ser 305 310 315 320 Arg ProPro Gln Val Pro Gly Asp Pro Glu Arg Ala His Met Asn Met 325 330 335 CysAsp Asp Leu Gly Gly Ile Val Tyr Lys Lys Lys Gln Leu Asp His 340 345 350Leu Lys Asn Leu Ala Asp Arg Leu Gly Val Ile Met Arg Leu Val Asp 355 360365 Glu Lys Pro Gln 370 8 400 PRT Caenorhabidits elegans 8 Met Asn LeuLeu Gln Arg Ala Leu Val Phe Thr Gly Gly His Ile Ser 1 5 10 15 Arg TyrGln Ala Val Ile Ala Val Asn Ser Val Gly Lys Asn Ala Arg 20 25 30 Phe TyrSer Thr Thr Asp Asp Asn Met Ala Ala Pro Glu Glu Ser Val 35 40 45 Val AlaLys Asp Glu Met Lys Arg Phe Met Val Glu Cys Met Thr Lys 50 55 60 Val GlyAla Thr Glu Ser His Ala Thr Gln Leu Ala Leu Val Leu Leu 65 70 75 80 GluGly Asp Ile Arg Gly His Tyr Ser His Gly Leu Asn Arg Leu Asp 85 90 95 MetTyr Val Arg Asp Ile Glu Gln Asn Val Cys Lys Gly Asp Gly Glu 100 105 110Pro Ile Ile Leu Lys Glu Lys Ala Gly Thr Ala Trp Val Asp Gly Asn 115 120125 Asn Leu Leu Gly Pro Val Val Gly Asn Phe Cys Met Asp Leu Ala Ile 130135 140 Glu Lys Ala Lys Asn Ala Gly Ile Gly Trp Val Val Ala Lys Gly Ser145 150 155 160 Asn His Tyr Gly Ile Ala Gly Trp Tyr Ala Leu Arg Ala MetLys Lys 165 170 175 Gly Met Leu Gly Met Ser Met Thr Asn Thr Ser Pro IleSer Phe Pro 180 185 190 Thr Arg Ser Ala Val Pro Ala Leu Gly Thr Asn ProIle Ser Leu Ala 195 200 205 Ala Pro Gly Thr Gly Asp Asp Ser Phe Val LeuAsp Met Ala Ser Thr 210 215 220 Thr Val Ala Ile Gly Lys Val Glu Leu AlaAla Arg Lys Glu Asn Pro 225 230 235 240 Val Pro Leu Ser Trp Gly Val GlyGlu Gly Gly Lys Glu Thr Thr Asp 245 250 255 Pro Thr Lys Val Leu Tyr GlyGly Gly Leu Leu Pro Leu Gly Gly Val 260 265 270 Glu Val Ser Gly Gly TyrLys Gly Tyr Gly Leu Ser Ser Met Ile Glu 275 280 285 Ile Phe Cys Gly IleLeu Ala Gly Ala His Trp Gly Pro His Val Arg 290 295 300 Lys Trp Met SerThr Lys Ser Glu Ala Asp Leu Gly Gln Cys Phe Val 305 310 315 320 Ala IleAsp Pro Glu Ala Phe Ala Pro Gly Phe Ala Asp Arg Leu Gln 325 330 335 AspPhe Met Gln Thr Met Arg Ala Leu Pro Thr Ser Ser Pro Ser Phe 340 345 350Lys Val Glu Val Ala Gly Asp Met Glu Arg Arg His Glu Ala Leu Val 355 360365 Glu Gln Leu Gly Gly Ile Pro Tyr His Lys Asn Gln Ile Thr Phe Val 370375 380 Asn Asp Leu Ala Ala Lys Leu Gly Val Lys Thr Val Asp Leu Val Gln385 390 395 400 9 22 DNA Artificial Sequence Vector polylinker primer 9gtaatacgac tcactatagg gc 22 10 20 DNA Artificial Sequence Vectorpolylinker primer 10 aattaaccct cactaaaggg 20 11 49 DNA ArtificialSequence Univeral primer to poly A tail 11 gagagagaga gagagagagaactagtctcg agtttttttt ttttttttt 49 12 18 DNA Artificial Sequence Primer12 agcaacaaaa cattggcc 18 13 18 DNA Artificial Sequence Primer 13ggcactgctt gggttgat 18 14 18 DNA Artificial Sequence Primer 14atcaacccaa gcagtgcc 18 15 21 DNA Artificial Sequence Primer 15cgattatggg ggcaatcaca c 21

What is claimed is:
 1. A purified polypeptide comprising an amino acidsequence that is at least 70% identical to the amino acid sequence ofSEQ ID NO:3. wherein the polypeptide has malate dehydrogenase activity.2. The purified polypeptide of claim 1, wherein the amino acid sequenceis at least 80% identical to the amino acid sequence of SEQ ID NO:3. 3.The purified polypeptide of claim 2, wherein the amino acid sequence isat least 90% identical to the amino acid sequence of SEQ ID NO:3.
 4. Thepurified polypeptide of claim 3, wherein the amino acid sequence is theamino acid sequence of SEQ ID NO:3.